Not Applicable.
This research is developing a synthetic method and in vivo designed reagent for the chemoselective, covalent modification of the phosphodiester group in nucleic acid polymers. Successful alkylation of phosphodiesters to form stable phosphotriesters has been accomplished through the development of chemistry based on the reactive paraquinone methide. The systematic analysis of model compounds is being used to add increasingly higher levels of selectivity to the alkylating methodology. The design of these model compounds is evolving to incorporate the necessary functionality to achieve the development of a final reagent which will be used for selective in vitro modification of a single phosphodiester group within a DNA or RNA target. This final reagent is designed to allow the delivery of a variety of reporter groups, drug agents or protein conjugates to DNA or RNA targets. This methodology would allow for the molecular level study of protein-nucleic acid interactions in such complex systems as the chromatin, where molecular level understanding is still very limited due to the complex, multi-protein machinery which operates at this level. This would also lead to an innovative approach for site-selective drug delivery to nucleic acid targets. Lastly, this methodology has the potential for transcription control through the site-selective delivery of transcriptional activators to genetic targets.
This proposal focuses on the synthesis and analysis of the model compounds and reactions which are being developed in the process of optimizing the reagent design. This research is providing a detailed understanding of various chemical reactions and molecular interactions. The knowledge gained and compounds produced in the process of this research are providing new synthetic methodology for nucleic acid chemistry and compounds for nucleic acid modification. This proposal describes the research necessary up to the total synthesis of a fully functionalized labeling reagent and its characterization.
1. Specific Aims
Rapid progress in sequencing the human genome1 opens new doors for potential technological developments for studying and treating disease at the foundational genetic level. One area of such potential development is the in vivo chemical modification of genomic DNA for diagnostics, therapeutics, and the study of biological processes. This requires progress in several areas of biomedical technology. Advances in oligonucleotide delivery to cells2 and sequence-specific recognition of DNA3 are two key areas. Our research program is targeting an unexplored area for the development of an innovative, chemical means to covalently deliver a variety of reporter groups, drug agents, or proteins to DNA. The ability to site-specifically attach such moieties to DNA would allow various genetic-based, biological studies to be conducted4 and provide a new means for efficient diagnostics5, therapeutics6 and biological control at the genetic level7.
Covalent modification of the phosphodiester group would be of most interest as it is the common, repeating, nucleophilic functional group throughout nucleic acid polymers. Whereas covalent modification of the nucleic acid bases generally leads to strand cleavage through depurination and will disrupt base pairing by interfering with hydrogen bonding, modification of the phosphate will likely have less effect on nucleic acid structure and function (FIG. 1).
This proposal will present the foundational research necessary for development of such a chemical reagent to accomplish this overall goal. This research is developing a variety of useful chemistry, synthetic methodology, and compounds in pursuit of the long-term goal.
The final in vivo designed reagent will covalently transfer attached molecules to a target phosphate group of a nucleic acid polymer (FIG. S1). The reagent is designed around a quinone methide with its bimodal electrophilic and a nucleophilic reactivity. The design features include:
An independently tethered delivering oligonucleotide and molecule to be transferred (1, loligo and R, respectively, FIG. S1). These are appended to a DNA synthesizer, machine-ready core reagent using standard automated, solid-phase, synthetic protocol for simplicity, efficiency and versatility.
Phosphate specificity through a guanidinium-phosphate complex (2). The guanidinium group will be substituted as needed to lower it nucleophilicity and prevent intramolecular reaction.
xe2x80x9cCagedxe2x80x9d reactivity initiated by proteolysis to afford the quinone methide precursor (3).
An intramolecular tertiary amine may be incorporated if it proves beneficial to assist 1,6-elimination8 to afford the intermediate quinone methide (4).
Alkylation of the phosphodiester with the quinone methide resulting in the in vitro release of the delivering oligonucleotide through lactonization to accomplish the transfer step (4 to 5). The oligonucleotide tether is designed to be cleaved at a slower rate than 1,6-elimination occurs. The intramolecular conjugate acid will afford stability to the trialkylphosphate prior to lactonization.
Formation of a covalently stable trialkylphosphate upon lactone formation by trapping the ailcylated product (5) and preventing reaction reversibility.
The reagent is an affinity transfer alkylating reagent (ATAR) for labeling the phosphodiester of nucleic acids. The research program is designed to streamline development of the ATAR by optimizing the chemistry through independent model system studies. The final reagent will be suitable for general use by attaching any delivering oligonucleotide on an automated synthesizer followed by attachment of a desired reporter group9, drug agent10, or protein conjugate11 on the solid support or post-synthetically. This provides a xe2x80x9cuser-friendlyxe2x80x9d reagent for use in modifying DNA, studying various nucleic acidprotein interactions, and for drug delivery applications.
The chemistry required for the ATAR is being developed through a variety of small molecule model studies. Each study requires minimal synthesis in order to independently investigate the various chemical aspects of the ATAR for optimization. The project has been designed so that the short-term model studies can be carried out by undergraduate research assistants. Progressively more complex studies are underway in order to coordinate the compatibility of the chemical reactions for optimal control of all aspects of the ATAR design. The chemistry necessary for the total syntheses of fully functionalized derivatives for incorporation into the ATAR on a DNA synthesizer is being developed in the course of these model studies. The multiple small component contributions required for this project make it an optimal training ground for short-term undergraduate research. This proposal maps out the investigations for development of the chemistry of the ATAR focusing on the model system syntheses and studies. Although the overall goal of this research is the envisioned applications of the ATAR mentioned above4-7, this will be beyond the time frame for which present funding is sought.
Several significant subset developments result from the pursuit of the overall research goal. One will be a simplified synthetic method12,13 for site selective alkylation and peralkylation of oligonucleotide phosphodiesters to produce trialkylphosphate modified oligonucleotides for various uses.14,15,5 This backbone modification affords enhanced hybridization properties16, antisense/antigene applications17,18 and peralkylations of the phosphodiesters will alter an oligonucleotides solubility properties for use in large-scale solution phase oligonucleotide synthesis. This research has already afforded a useful synthetic method for modifying phosphodiesters with the formation, isolation and fall characterization of trialkylphosphates19. Some aspects of commercial potential for this methodology are being pursued with industrial support20. A non-specific chemical nuclease is being developed in conjunction with this research and a method for the site-selective hydrolytic cleavage of DNA21,22.
2. Background and Significance
Heterobifunctional crosslinking reagents. containing a cleavable linker have been developed for studying protein-protein interactions23. These reagents require an invasive chemical step to transfer the probe molecule from the delivering species to the target protein. This limits their use to in vitro applications. The ATAR we are developing will involve an in vitro cleavage step of the initially formed crosslinked complex in order to release the delivering oligonucleotide. This forms a DNA target which has been covalently modified with a small molecule carrying the independently attached label. The independent synthetic attachment of both the delivering oligonucleotide and the desired reporter group, drug agent, or protein provides a versatile ATAR for various applications.
One example of a reagent which transfers a methyl group from a nucleic acid binding reagent to a nucleic acid base in vitro comes from the work of Gold and coworkers24. They produced a methylating reagent by tethering methyl sulfonates to a dipeptide lexitropsin, an A/T-rich minor groove binder. This reagent allowed methylation of the adenine-N3 selectively, resulting in the release of the lexitropsin sulfonic acid byproduct. The goal of this proposal is to define a method to extend this type of in vitro transfer chemistry beyond methylation to the transfer of a large variety of reporter groups or drug molecules. This will be accomplished by having them tethered to a reactive quinone methide25,26 which will initiate nucleophilic attack followed by the in vitro release of the delivering molecule. Further, the ATAR being developed will be latently reactive upon photolysis after binding to the target site in order to minimize secondary alkylation reactions. It will also target the phosphate residue of nucleic acids in order to minimize perturbation of the bases, leaving the nucleic acid free for hybridization.
Although the phosphate residue of nucleic acids is not the chemoselective site for alkylation by many routinely used electrophilic reagents27, in vitro ailcylation of the phosphodiester to afford phosphotriesters is observed. Ethylnitrosourea (GNU) shows the highest selectivity for phosphotriester formation relative to methylnitrosourea (MNU), dimethylsulfate (DMS) and ethylmethanesulfonate28. Expressed in terms of total DNA alkylation, the extent of phosphodiester alkylation by ENU has been estimated to be between 59%28a and 70%28c.
A quinone methide is an effective ailcylating agent with a dialkylphosphate (see Preliminary Results). A quinone methide (FIG. 2) is a potent electrophile due to its highly polarized nature. Rearomatization of the quinone methide ring is a strong driving force for reaction29. This relatively hard electrophile is a good alkylating agent for the hard phosphate oxygen30.
Skibo and coworkers have recently developed a molecule (5, FIG. 3) that alkylates the phosphate residue of nucleic acids31. This molecule contains a binding region which recognizes the adenine-thymine (A-T) base pair (and to a lesser extent the guanine-cytosine (G-C) base pair). The alkylating region is composed of an aziridium moiety for selective phosphate alkylation (6, FIG. 3) instead of normal alkylation of N-732.
Day and coworkers attempted to develop a reagent for alkylation of DNA phosphate groups using para-bromomethylbeazoyl choline iodide33. Unfortunately, it was later reported the reagent was polymerizing and phosphate alkylation was not occurring34. This work suggests the challenge in finding a strong enough electrophile to selectively react with a phosphate. As indicated, we have already shown that quinone methides alkylate dialkylphosphates in an aqueous environment.
The guanidinium functional group is extensively used in biological systems and various artificial receptors for phosphate recognition and binding35. This type of ionic association of cationic amine residues with DNA has been successfully used by other researchers in order to enhance binding to DNA36. As charge-charge attractions are the strongest noncovalent molecular interactions, salt bridges between nucleic acid phosphates and positively charged amino acid side chains are individually the highest strength interactions in protein-nucleic acid interactions37.
The ATAR we are developing takes advantage of this type of guanidinium-phosphate ionic association to direct the alkylation process. The precursor to the quinone methide will incorporate a guanidinium residue to enhance the effective concentration of the phosphodiester. The guanidinium group may associate with other nucleic acid sites, such as the bases38; however, the thermodynamic preference for two point hydrogen bonding and charge pairing of a guanidinium-phosphate complex is well accepted35,37.
3. Preliminary Results
Various model system studies are being conducted to develop the chemistry necessary for the ATAR. Below are nine key results which contribute to the ATAR development. In the area of in vivo design, the importance of a compound comes only with proven function. This of ten delays publication of foundational work until the significance of the chemistry is verified. The formation of isolated, fully characterized trialkylphosphates has been accomplished to provide a useful synthetic approach for modifying phosphodiesters. Due to this recent demonstration of function, publication of these results are in progress19 and publication of the foundational work which supported it will follow39. There is presently a provisional patent covering many of these developments40. Some aspects of commercial potential of the phosphotriester forming reactions are being pursued with industrial support20.
The small scale of these model studies as individual components of the projects overall goals make the research readily suited to undergraduate research. In less than two years of effort on this project, five different undergraduate and three graduate students have been involved at different times.
3.1. Quinone methide alkylation of a phosphodiester to form a phosphotriester. Studies of a quinone methide with a dialkylphosphate have been conducted39a 2,4,6-Trimethylphenol was quantitatively converted to quinone methide 7 with AgzO41 and dibenzylphosphoric acid was added to produce phosphotriester 8 as the exclusive product (FIG. S2)41.
3.2. Phosphotriester product is favored upon protonation. Formation of 8 is an equilibrium process. Trialkylphosphate 8 is favored under acidic conditions which protonate the quinone methide oxygen leading to the phenol. However, under basic conditions where the phenol is deprotonated or conditions acidic enough to protonate the phosphotriester oxygen, 7 is favored39a. As initially seen by the effect of various acids in the pKa range shown in FIG. S3, this should favor phosphotriester formation under biologically relevant conditions near pH 7.
3.3 Kinetic favorability of phosphotriester formation over hydrolysis in the presence of water. The reaction of quinone methide 7 and two equivalents of dibenzylphosphoric acid in the presence of excess water (xcx9c200 equivalents for a homogeneous solution) afforded only trialkylphosphate 8 as the product in the equilibrium by 1H NMR analysis. Minor amounts of the benzyl alcohol hydrolysis product was evident by 1H NMIR analysis after 18 hours at ambient temperature. Trialkylphosphate 8 is the kinetic product. A similar amount of 8 is produced in the presence of a much higher concentration of water (3,000 equivalents forming a bilayer) at ambient temperature after 30 minutes. However, the benzyl alcohol hydration product begins to drain off the kinetic ally formed 8 affording complete conversion to benzyl alcohol after 18 hours. Hydrolysis to benzyl alcohol appears to be the thermodynamic product39. Similar results of quinone methides reacting with amino acid derivatives under aqueous conditions have been reported by other researchers43.
3.4. Hydrolytic stability of an acetylated trialkylphosphate derivative. The effect of protonation on the alkylation reaction above, and the trialkylphosphate being the kinetic product, suggested trapping of the phosphate alkylated DNA as lactone derivative 4 (FIG. S1) should be favored over hydrolysis (to afford a benzyl alcohol) under physiological òconditions. Investigation of the stability of lactone trapped trialkylphosphate product was the next step. The high stability of independently synthesized 944 (FIG. 4) in water (pH 6.5, 40xc2x0 C., overnight) demonstrates the expected stability of the lactone product 4 (FIG. S1) which will result from the ATAR phosphate alkylation reaction39b.
3.5. Trapping stable phosphotriesters through tandem lactonization after quinone methide alkylation of a phosphodiester. The isolation of stable, fully characterized products has been imperative to the development of useful synthetic methodology from this research. This has now been accomplished19. Variety of ester derivatives have been synthesized to study the requirements for trapping the trialkylphosphate through lactonization. Characterizable quinone methide intermediates are prepared via Ag2O or PbO2 oxidation. It proved necessary to synthesize derivatives with an oxygen at the ortho-position (catechol derivatives, 10a, FIG. 5) to exclude the formation of ortho-quinone methides upon oxidation if the esters were tethered through a methylene at the ortho-position (10b, R2=H, H, FIG. 5). Attempts at making secondary or tertiary substituted tethers at the ortho-position (10b, R2=H, CH3 or CH3, CH3, FIG. 5) resulted in facile lactonization (gem-dialkyl effect) circumventing oxidation to the quinone methide. These catechol derivatives may provide a beneficial modification to the ATAR design. An oxygen at the ortho-position of a para quinone methide appears to increase the reactivity of the quinone methide towards nucleophilic addition45,46. The ortho-oxygen is expected to affect the rate of the lactonization reaction and the stability of the lactone product towards hydrolysis. This catechol-type system (10a) will be compared to the corresponding phenol system with an ortho-alkyl tether (10b). The quinone methide from the latter systems will be formed by 1,6-elimination of a benzylic leaving group.
The ester derivatives were made in five steps from 2,4-dimethylphenol47. These esters include three classes: high, intermediate and low reactive derivatives (see FIG. S3). The high reactive esters lactonized to afford 12 under the mildly basic conditions of oxidation. The low reactive derivatives were oxidized to the corresponding quinone methide 13 and underwent dibenzylphosphate addition; however, were not able to lactonize under the alkylation conditions. The intermediate reactive derivatives were successfully oxidized to para-quinone methide intermediate 13, alkylated the dibenzylphosplate to 14, and lactonized to afford trapped trialkylphosphate product 15 (FIG. S3).
This now provides a useful synthetic approach towards the covalent functionalization of phosphodiesters. The key, fundamental reactions of the designed ATAR reagent have been successfully demonstrated. The details of these investigations are being submitted for publications1 9. The alkylation of nucleotide derivatives are presently being studied having obtained enough of various required dinucleotides for NMR analysis of the phosphodiester alkylation48.
119 
3.6. Quinone methide formation through photolytic-initiated 1,6-elimination followed by phosphodiester alkylation. After significant effort, the conditions necessary for photolytic removal of a protecting group followed by 1,6-elimination to afford the para-quinone methide and reaction with dibenzylphosphate has been accomplished. Although there are numerous reports of reactions which occur through the presumed formation of quinone methide intermediates by 1,6-elimination processes26,49,50 to our knowledge this is the first case of caged, photolytically-activated p-quinone methide formation via elimination with characterization39c,51. Multiple derivatives have been synthesized as discussed below (point 8, FIG. T2). The first successful reaction was accomplished using 16 (FIG. S4). Photolysis of 16 (150W xenon arc lamp, BiCI3/HCl filter, S2 ambient) was monitored by 1H NMR in CDCl3 with either: (A) one equivalent of Agxcx9c(BnO)2POxcx9c-salt, or (B) one equivalent of (i-Pr)2EtNHxcx9c(BnO)2PO2-salt. After photolysis for one hour, nearly all of 16 was deprotected to form a mixture of phenol 17, quinone methide 7 and trialkylphosphate 8 in approximately 2:1:1 ratio, respectively. This photolytic-initiated reaction did not go to completion, but appears to form an equilibrium mixture of 17:7:8 under these conditions. Note that under aqueous conditions for which the ATAR is being designed to operate, the chloride will not be a competitive nucleophile, and will rapidly diffuse away from the quinone methide. Preliminary experiments having water present show no sign of equilibrium back to 17. This experiment did not have the benefit of an intramolecular trap to drain off the kinetic preferred trialkylphosphate or the assistance of a phosphate-directing guanidinium group.
3.7. Hydrolytic stability of the quinone methide precursor and photolytic stability of the quinone methide intermediate and the trialkylphosphate product. Experiments have demonstrated the benefit of a carbonate protected phenol (e.g., 16, FIG. S4) for greatly increasing the stability of the benzylchloride. Hydrolysis of quinone methide precursors has been a problem with many quinone methide-based, biologically reactive molecules.26,43,49 The carbonate protected 16 has shown no sign of hydrolysis at 250xc2x0 C. in 33% D20/CD3CN for two days. Related benzyl protected derivatives hydrolyze relatively rapidly39.
Due to the precedent for photolytic-induced homolytic reactions occurring with derivatives containing benzylic leaving groups, 53 and the appearance of various byproducts in earlier reactions attempted, an investigation of the photolytic stability of the intermediate quinone methide and the triallcylphosphate product was undertaken. Pre-formed quinone methide 7 was photolyzed under conditions used to afford 8 above (FIG. S4), and no reaction was evident by 1H NMR analysis after 3 hours. Similarly, trialkylphosphate 8 was photolyzed under the same conditions and showed no sign of reaction after 3 hours.
3.8. Studies of various combinations of photolabile protecting groups with different benzylic leaving groups for quinone methide formation. Successful conditions for producing identifiable quinone methide derivatives through photolytic-initiated 1,6-elimination reactions have now been realized39C. This led to the synthesis of derivatives containing either the o-nitrobeazyl (NB), the a-methyl-3,4-dimethoxy-2-nitrobenzylcarbonate (DMNBC) or the dimethoxybenzoin carbonate (DMBC) protecting group54,55 with a variety of leaving groups at the benzylic position (FIG. T2)56. These derivatives are being examined to determine effects of the different substituents on their hydrolytic stability39c,43, the rates of quinone methide formation and the alkylation reaction rates45,49. These particular derivatives allow correlation with other systems being used to study the quinone methide ailcylation reaction and the lactonization reaction.
3.9. Competitive guanidine cydlization: Phosphotriester formation with a quinone methide and a phosphodiester-ethylguanidinium salt. A possible guanidine 5-exo-trig cyclization on the quinone methide of the designed ATAR was realized. This potential competition is under examination and approaches to prevent it, if necessary, are being developed (see 4.1). Initial results57 and a thorough literature search suggest this may be negligible58,59. The initial analysis looked at the effect of ethylguanidinium on the dibenzylphosphate alkylation reaction with quinone methide 7. As shown in FIG. S5, 0.5 equivalents of ethylguanidiniumdibenzylphosphate salt (18) in 8:1 DMSO-d6/D20 was added to a solution of quinone methide 7 in CDCl3. Due to solubility problems, NMR integration shows approximately 0.3 equivalents of 18 remained in solution. Within 30 minutes at ambient temperature, the same equilibrium of 7:8 was apparent which was formed without the ethylguanidinium present (i.e., 2:1 of 7:8, FIG. S2). Normalizing the reaction to the total amount of 18 present (0.3 equiv.), a 2:1 ratio of the equilibrium mixture of quinone methide 7 to trialkylphosphate 8 appears as an overall 10% formation of 8 by 1H NMR integration. Again, the presence of D20 had no effect on the kinetic formation of 8; however, with no intramolecular trap to drain off the kinetically formed 8, over the next several hours the presence of benzyl alcohol increased. At no point in the reaction was there any evidence of the ethyl guanidine adding to the quinone methide.60,61 
Although this experiment examined an intermolecular reaction and the competitive cyclization reaction in the ATAR will be intramolecular, it should be realized that there was a 1:1 ratio of the phosphate and guanidine present for reaction with the quinone methide in this experiment. In the ATAR, the complexation of the guanidinium with the phosphodiester will similarly result in a 1:1 ratio of the two components in proximity of the quinone methide. Effects of hydrogen bonding lowering the nucleophihicity of the phosphate while increasing the nucleophilicity of the guanidine were present equally in the above experiment as they will be in the ATAR-DNA alkylation reaction so these effects would also still result in the expected preference for phosphodiester alkylation.
4. Research Design and Methods
Completion of the model studies described above and those following will accomplish the design optimization of the individual ATAR components. Studies with increasingly more complex systems are beginning to coordinate the reactions into a functional derivative.
4.1. Investigations of quinone methides with tethered guanidine functional groups. Model systems are being investigated to determine the effect of a tethered guanidinium in directing the phosphodiester alkylation reaction. Our initial efforts have focused on the use of two commercially available amines tyramine and octopamine, which were converted to their respective guanidinium-phosphate salt derivatives 18 and 19 (FIG. 662. Oxidation of 18 (and abis-Boc guanidine derivative) has not succeeded using Ag2O, PbO2, or DDQ. The inability to oxidize p-cresol suggests the 2,6-dimethyl derivative may be necessary. We recently accomplished a Stille coupling in a related 2,6-diiododerivative63 and are preparing 20 for quinone methide formation through oxidation64a. A separate route to synthesize 20 has been developed by an undergraduate researcher and is in its final step for completion64b. The 1,6-elimination of unactivated benzyl alcohol 19 to afford quinone methide has yet to succeed using acidic thermolysis. Derivative 21 is being prepared for the more facile 1,6-elimination to form the quinone methide64c.
The experiment reported above (FIG. S5) suggests that cyclization of guanidine on the quinone methide may not occur with the guanidiium-phosphate salt complex. Should this occur, adjusting the nucleophilicity of the guanidine should alleviate this possible competition. Based on reported pKa values for various substituted guanidines 8,65 a phenyl guanidine (10.8) is sufficiently less basic than a methyl guanidine (14.1). The nucleophilicity should be similarly weakened while still favoring the guanidinium form. Phenyl-substituted guanidines 20 and 21 (Rxe2x80x3xe2x80x2=Ph, FIG. 6) are being synthesized5 to examine this effect.67 
4.2. Incorporation of a guanidinium group and the ortho-tethered ester for lactonization. The synthesis of a derivative which will examine the effect of both the guanidinium group for phosphate specificity and the ortho-tethered ester for lactonization to trap the trialkylphosphate is being synthesized according to FIG. S6. Although results since the initiation of this synthesis may require changes in the protecting group strategy,68 all of the reactions have already been worked out in other model systems so the completion of this derivative is expected without complications.
Examination of this model system in reactions with phosphodiesters will allow determination of the concerted efficiency of the guanidinium-phosphate complexation for alkylation specificity and trapping of the trialkylphosphate as the lactone for product stabilization. Note that the methyl ester will initially be attempted as the lactonization is expected to be more efficient with the all carbon tether as opposed to the catechol system which has already been optimized (see section 3.5)19. If necessary, the lactonization rate can be increased through Heck reaction with methyl-methacrylate (FIG. S6, step four). This will place methyl groups on the ester tether and increase the rate of lactonization.69 
4.3. Incorporating a proton shuttle into the ATAR. Some preliminary experiments suggest incorporating a tertiary methylamine into the tether ortho to the phenol may help facilitate the ATAR reactivity. Having an estimated pKa of 9.8,8 this will act as a proton shuttle to assist quinone methide formation through deprotonation for 1,6-elimination, the conjugate acid will help to activate the quinone methide through reprotonation, and it will assist in the lactonization reaction by deprotonation of the phenol. This will have little competition with guanidine protonation, so is expected to show no deleterious effects. Analysis of this design feature will be investigated in model systems incorporating this modification. These will be synthesized using methods shown in the total syntheses below. If this modification proves unnecessary, the total syntheses below will be simplified, but will be shown with the amine to exemplify the more challenging approach.
4.4. Positioning of the guanidine group for optimal phosphate recognition and quinone methide alkylation. An additional set of model studies will be carried out if proven necessary based on the initial system being investigated (system A, FIG. 7). The three systems are shown in FIG. 7 based on the position of the guanidiium group. Note that each system has at least one tautomer where phosphate addition will be more favored.70 The flexible, non-static nature of these non-covalent interactions should be realized.
Model system A (FIG. 7) will be examined through the studies described above. Analysis of systems B and C (FIG. 7) will be carried out if there are deficiencies in system A. The synthesis of model systems B and C will be readily accomplished by modification of the synthetic approaches described below (sections 4.5, B and C). The synthesis of these model systems will therefore not be described separately, as the formation of simplified derivatives to examine the guanidine position effect can readily be seen from examination of the total syntheses which will be described below.
4.5. Syntheses of functionalized ATAR model systems. Much of the chemistry for synthesizing ATAR derivatives is being developed through the various model system studies. Three different fully functionalized DNA-synthesizer machine-ready ATAR derivatives may be synthesized. These are described below. Note that not all of these systems will be necessary. The model studies described above will define which system will be optimal and whether or not the tethered proton shuttle will be beneficial. The syntheses described below will produce the most difficult ATAR model systems. The system which may be most optimal based on model studies should therefore be no more difficult than any one of the syntheses described below. Should the incorporation of a proton shuttle prove to be of no benefit, then each of the syntheses below will be readily simplified.
(A) An ATAR derivative with the guanidine at the exocydic methylene of the quinone niethide. DNA synthesizer machine-ready derivative 25 will be prepared in ten steps from octopamine (22, FIG. S7). Most of the key steps have already proven effective in model studies. The synthesis involves a Rathke guanylation,55 and ortho-bromination71 followed by iodination72 to make 23 for the Stile allylation selectively with the more reactive aryliodide.73 This will be converted to the amine through ozonolysis74 and reductive amination.75 The Heck reaction with acrylic acid will afford 24.76 The diimide reduction77 for reducing the alkene has already been accomplished in a model study without affecting the nitrobenzyl group.
(B) An ATAR derivative with a meta-benzylic guanidine substituent. A machine-ready derivative having the guanidine at the meta-benzylic position will be synthesized using a more convergent approach with a Diels-Alder reaction as the key step. A highly functionalized Danishefskytype diene78 will be synthesized in four steps from the dianion of acetoacetic amide 26 (FIG. S8)79. Dianion akylation80 will afford 27 which will be reduced to the aldehyde81 and converted to diene 28 by the standard approach.82 
There is good precedent for the success of the cycloaddition of diene 28 with allene 29 to produce phenol 30 (FIG. S9).83 Phenol 30 will be converted to machine-ready derivative 35 in 11 steps. Resonance effect allows for the selective hydrolysis of the benzylic methyl ester of 30.84 A tandem Curtius rearrangement-Rathke reaction with in vitro trapping of the amine as the protected guanidine66 will be examined.85 The B-trimethylsilylethanesulfonyl (SES) protected guanidine86 will be stable throughout the synthesis, but cleaved with fluoride ion after solid-phase synthesis without hydrolyzing the carbonate (MDNB) group on the phenol. The final conversion of the methyl-trimethylsulfide to the carboxylic acid will be accomplished as in Schreiber""s total synthesis of cyclotheonamide B with related functionality in the molecule.87 
(C) An ATAR derivative with a meta-guanidine substituent. If validated in model studies, a machine-ready derivative having the guanidine directly on the benzene ring will be prepared by one of two approaches. (1) A simple modification of the above approach using methyl acetylenedicarboxylate as the dienophile.82 (2) A directed ortho-lithiation starting from 4-amino-salicylic acid taking advantage of a MEM88 and Boc89 protecting group to assure regioselectivity to 37 (FIG. S10).90 Most of the other steps are repetitive of those in the above syntheses or readily carried out using standard chemistry.
4.6. Evaluation with nucleotide oligomers. The following studies will be incorporated as time permits during the requested finding period. These investigations will be discussed in chronological order, and will be accomplished to the extent allowable during this time frame.
4.6.1. Nucleotide alkylation studies. Prior to attaching the phosphodiester alkylating reagents to oligonucleotides for site-selective delivery, reactions will be run using the appropriate derivative synthesized above91 with dinucleotides for complete product characterization and reaction optimization.48,92 Dodecanucleotides will then be examined for peralkylation. The presence of the guanidinium groups in the alkylated polymer will maintain the water solubility of the trialkylphosphate product.93 The reversal of charge will reverse the polarity necessary for PAGE analysis or slow the migration of partially alkylated oligonucleotides.36 Assessing the degree of alkylation of the whole oligonucleotides can be determined qualitatively by gel migration analyses using PAGE on the 5xe2x80x2-32P labeled oligos.94,95,96 Initial digestion of the oligonucleotides from the alkylation reaction with snake venom phosphodiesterase and/or calf intestine alkaline phosphatase will result in cleavage of the oligonucleotides only at unmodified phosphodiester linkages, as phosphotriester linkages are known to be stable to degradation.16a HPLC analysis of the resulting products for the degree of allcylation will assess if there was any regioselectivity.97 The degree of alkylation will be further analyzed by high resolution mass spectrometry (MALDI-TOF). NMR analysis will be attempted to determine chemo- and possibly diastereo-selectivity""s and for assessing the structural characteristics of the products.98 Crystallization of the products for x-ray diffraction analysis may succeed with the guanidinium group incorporated.
4.6.2. Oligonucleotide attachment. The delivery oligonucleotide of desired sequence will be synthesized on an automated DNA synthesizer according to standard protocol.99 Modifying oligonucleotides with any desired linker is common practice17d,e,g,h,i. A C12 chain length phosphoramidite will be synthesized100 and attached to the oligonucleotide99,101. A standard esterification reaction will attach the ATAR derivative as synthesized above to the linker-OH on the solid support102. Mild alkaline hydrolysis of the TFA-protected amine103 will allow the attachment of the desired reporter group, drug agent, or protein conjugate. Some examples of reporter groups for initial studies of oligonucleotide modification using this ATAR include: fluorescein-5-isothiocyanate,104 the EDTA-Fe(TI) moiety105, and tris(2,2xe2x80x2-bipyridine) ruthenium(II) (Ru(bpy)32+106. Complete protecting group removal and cleavage from the solid-support107 will afford a functional ATAR108. Drugs6,10 and derivatized proteins7,11 will be attached similarly. The resulting ATAR derivatives will be purified by HPLC. The derivatized oligonucleotides will be characterized by enzymatic digestion and HPLC analysis against coinjections of standard solutions of the nucleoside components and a reagent standard with the attached linker. An exact mass will also be obtained. ATAR attachment will be confirmed by UV analysis.109 
Affinity cleavage experiments can be conducted with the EDTA-e(II) group attached to the ATAR for analysis of labeling both single- and double-strand target DNA according to established methods3,110. Analysis of the diffusible cleavage pattern on the DNA to which the ATAR has been delivered will allow assessment of the structural characteristics of the ATAR-DNA interactions.
4.7. Future studies. The ability to modify nucleosomal DNA will allow various crosslinking and autocleavage investigations to be conducted for enhancing our understanding of DNA-protein interactions in the chromatin4. Transcriptional regulation will be studied by using the ATAR to attach transcriptional regulator GCN5p7 and other transcriptional activators to selected sites on DNA7b. It will also be of interest to study how drugs known to bind to, and react with DNA will be affected by their covalent attachment through the ATAR.111 
5. Human Subjects: None
6. Vertebrate Animals: None
7. Literature Cited
(1) For a complete resource covering many aspects of the human genome see the Genome Database (GDB) hosted at Johns Hopkins University (http://gdbwww.gdb.orgl): Fasman, K. H.; Letovsky, S. I., Li, P.; Cottingham, R. W.; Kingsbury, D. T. xe2x80x9cThe GDB Human Genome Database Anno 1997,xe2x80x9d Nucleic Acids Res. 1997, 25, 72-80.
(2)
(a) Leonetti, J. P.; Degols, G.; Clarenc, J. P.; Mechti, N.; Lebleu, B. xe2x80x9cCell Delivery and Mechanism of Action of Antisense Oligonucleotides,xe2x80x9d Prog. Nucleic Acids Res. Mol. Bid. 1993, 44, 143-66.
(b) Zon, G. xe2x80x9cBrief Overview of Control of Genetic Expression by Antisense Oligonucleotides and In Vivo Applications,xe2x80x9d Molec. Neurobiol. 1995, 10, 219-29.
(3)
(a) Thuong, N. G.; Hxc3xa9lxc3xa9ne, C. xe2x80x9cSequence-Specific Recognition and Modification of DoubleHelical DNA by Oligonucleotides,xe2x80x9d Angew. Chem. Int. Ed. EngI. 1993, 32, 666-90.
(b) Dervan, P. B. xe2x80x9cReagents for the Site-Specific Cleavage of Megabase DNA,xe2x80x9d Nature 1992, 359, 87.
(c) Dervan, P. B. xe2x80x9cDesign of Sequence Specific DNA Binding Molecules,xe2x80x9d Science 1986, 232, 464.
(4) A system such as being proposed will be particularly valuable for providing information at the molecular level in multi-protein complexes interacting with nucleic acids. These would include the complex protein-nucleic acid interactions of the chromatin involved in chromosome condensation-decondensation, DNA replication, transcription, transcription regulation and DNA repair. Molecular level details in such complex systems are difficult to achieve by existing biochemical techniques and advances in molecular biology require innovative approaches to begin to develop a more thorough molecular level understanding of the chromosomal protein machinery. For example, the presumed role of histone H 1 in transcriptional repression might be studied by site-specifically modifying a target DNA binding sequence with crosslinking and redox activated cleaving functionality for mapping DNA-histone H1 interactions:
(a) Paranjape, S. M.; Kamakaka, R. T.; Kadonaga, J. T. xe2x80x9cRole of Chromatin Structure in the Regulation of Transcription by RNA Polymerase II,xe2x80x9d Annu. Rev. Biochem. 1994, 63, 265-97.
(b) Felsenfeld, G. xe2x80x9cChromatin as an Essential Part of the Transcriptional Mechanism,xe2x80x9d Nature 1992, 355, 219-24.
(c) Halmer, L.; Gruss, C. xe2x80x9cInfluence of Histone H1 on the in vitro Replication of DNA and Chromatin,xe2x80x9d Nucleic Acids Res. 1995, 23, 773-78.
(5) The ability to label a hybridization-recognized sequence of DNA should afford an efficient approach to genetic diagnostics from blood samples. The chemistry being developed will allow the efficient synthesis of multiply-labeled oligonucleotides which can be used for genetic diagnostics by methods such as fluorescence in-situ hybridization (FISH).
(a) Brenner, M.; Dunlay, T. xe2x80x9cFluorescence In vitro Hybridization. Hardware and Software Implications in the Research Laboratory,xe2x80x9d Amer. Laboratory 1995, 55-58.
(b) For lanthanide-labeled DNA probes, see: Lxc3x6vgren, T.; Hurskainen, P.; Dahlxc3xa9n, P. in Nonisotopic DNA Probe Techniques, Kricka, L. J., Ed.; Academic Press, Inc.: San Diego; 1992, pp. 227-274.
(6) A particularly appealing application would be in the area of site-specific drug delivery to genetic targets. The non-specific deliterious effects of chemotherapy on healthy cells could be alleviated using such a system to covalently deliver an antitumor antibiotic directly to a target DNA sequence.
(7) Innovative experiments which could be attempted with such a system include modifying nucleosomal DNA with transcriptional regulator GCN5p. This transcriptional regulator functions as a complex with two other proteins (ADA2p and,ADA3p). It has recently been found to be a histone acetyltransferase. Histone hyperacetylation is thought to facilitate transcription by chromatin disruption, but it is not clear whether the hi stone hyperacetylation is a result of chromatin disruption during the transcription process, or an initiator. This regulatory complex with GCN5p is recruited to a specific gene through interactions with other DNA binding transcription factors. A system such as being proposed would allow site-specific delivery of this regulatory protein to a particular chromatin site. This could then recruit the regulatory complex and other transcription factors and thereby initiate transcription of a selective gene. Obviously, such an approach could be used to regulate many cellular functions through selective control of genetic transcription. For leading references see:
(a) Wolffe, A. P.; Pruss, D. xe2x80x9cTargeting Chromatin Disruption: Transcription Regulatorg that Acetylate Histones,xe2x80x9d Cell 1996, 84, 817-19.
(b) Ptashne, M.; Gann, A. xe2x80x9cTranscriptional activation by recruitment,xe2x80x9d Nature 1997, 386, 569-77.
(8) Although only a very crude measure, estimated pKa values calculated from the effects of various related substituted derivatives suggest that the proton shuttle processes proposed should occur as drawn in scheme 1. The relevant pKa values (H2O, 25xc2x0 C.) include: 2,4,6-trimethylphenol (10.88), 3-aminophenol (9.83), m-cresol (10.00), phenol (9.99), Et2MeN (10.4), phenethylamine (9.83), ethylamine (10.63), guanidine (14.38), methylguanidine (14.1), phenylguanidine (10.77). From these values, pKa estimates in the ATAR may be approximated assuming additivity of substituent effects. The approximated pKa would be: 10.7 for the phenoxide with a 3-amino group on the ring (the effect of a 3-amino on the pKa of phenol is ApKa=xe2x88x920.16; thus approximating from the pKa of 2,4,6-trimethylphenol=10.88-0.16=10.7, other values are determined in a similar way), 10.9 for the phenoxide with the 3beazylic-amino group, 9.8 for the tertiary amine, 10.8 for the guanidine directly substituted on the arene ring, 10.5 for the benzylic guanidine with a phenyl substituent. The pKa values are from Lange""s Handbook of Chemistry, Dean, J. A, Ed.; McGraw-Hill: N.Y. 1992, 14th edition.
(9) Reporter groups would include fluorescent probes (For example, see: Haugland, R. P. Molecular Probes: Handbook of Fluorescent Probes and Research Chemicals, Larison, K. D., Ed.; 1995-1997, 6th Edition, Molecular Probes, Inc., Eugene, Oreg.), probes used for recognition of a specific species such as biotin/avidin and antibodies, luminescent probes, probes which are chemically or redox reactive, radionuclear probes, and magnetic moieties.
(a) Wilbur, D. S. xe2x80x9cRadiohalogenation of Proteins: An Overview of Radionuclides, Labeling Methods, and Reagents for Conjugate Labeling,xe2x80x9d Bioconjugate Chem. 1992, 3, 433-471.
(b) Peters, K.; Richards, F. M. xe2x80x9cChemical Cross-linking: Reagents and Problems in Studies of Membrane Structure,xe2x80x9d Ann. Rev. Biochem. 1977, 46, 523-51.
(c) Ji, T. H. xe2x80x9cThe Application of Chemical Cross linking for Studies on Cell Membranes and the Identification of Surface Reporterg,xe2x80x9d Biochim. Biophys. Acta 1979, 559, 39-69.).
(10) For examples of drugs which could be readily attached, see a listing of anticancer agents along with associated references in: Calbiochem Biochemical and Immunochemical 1996/97 Catalog, p. 539, San Diego, Calif.
(11) For an example of protein conjugation to an oligonucleotide for directing nuclease activity, see: Pei, D.; Corey, D. R.; Schultz, P. G. xe2x80x9cSite-specific Cleavage of Duplex DNA by a Semi-synthetic Nuclease via Triple-helix Formation,xe2x80x9d Proc. Nat. Acad. Sci. USA 1990, 87, 9858.
(12) For conventional synthesis of phosphate modified oligonucleotides, see:
(a) Hayakawa, Y.; Hirose, M.; Hayakawa, M.; Noyori, R. xe2x80x9cGeneral Synthesis and Binding Affinity of Position-Selective Phosphonodiester- and Phosphotriester-Incorporated Oligodeoxyribonucleotides,xe2x80x9d J. Org. Chem. 1995, 60, 925-30.
(b) Hayakawa, Y.; Wakabayashi, S.; Kato, H.; Noyori, R. xe2x80x9cThe Allylic Protection Method in Solid-Phase Oligonucleotide Synthesis. An Efficient Preparation of Solid-Anchored DNA Oligomers,xe2x80x9d J. Am. Chem. Soc. 1990, 112, 1691-96.
(c) Kuijpers, W. H. A.; Huskens, J.; Koole, L. H.; Van Boeckel, C. A. A. xe2x80x9cSynthesis of Well-Defined Phosphate-Methylated DNA Fragments: the Application of Potassium Carbonate in Methanol as Deprotecting Reagent,xe2x80x9d Nucleic Acids Res. 1990, 18, 5197-205.
(d) Alul, R. H.; Singman, C. N.; Zhang, G.; Letsinger, R. L. xe2x80x9cOxalyl-CPG: A Labile Support for the Synthesis of Sensitive Oligonucleotide Derivatives,xe2x80x9d Nucleic Acids Res. 1991, 19, 1527-32.
(e) Froehler, B. C. xe2x80x9cDeoxynucleoside H-Phosphonate Diester Intermediates in the Synthesis of Internucleotide Phosphate Analogues,xe2x80x9d Tetrahedron Let. 1986, 27, 5575-78.
(13) For additional synthesis reviews see:
(a) Beaucage, S. L.; Iyer, R. P. xe2x80x9cAdvances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach,xe2x80x9d Tetrahedron 1992, 48, 2223-2311.
(b) Hobbs, J. B. xe2x80x9cNucleotides and Nucleic Acids,xe2x80x9d Organophosphorus Chemistry 1990, 21, 201-321.
(c) Sonveaux, E. xe2x80x9cThe Organic Chemistry Underlying DNA Synthesis,xe2x80x9d Bioorg. Chem. 1986, 14, 274-325.
(14) Studies using phosphate triester modified oligos for duplex structure studies with RNA and DNA:
(a) Letsinger, R. L.; Bach, S. A.; Eadie, J. S. xe2x80x9cEffects of Pendant Groups at Phosphorus on Binding Properties of d-ApA Analogues,xe2x80x9d Nucleic Acids Res. 1986, 14, 3487-99.
(b) Summers, M. F.; Powell, C.; Egan, W.; Byrd, R. A.; Wilson, W. D.; Zon, G. xe2x80x9cAlkyl Phosphotriester Modified Oligodeoxyribonucleotides. VI. NMR and UV Spectroscopic Studies of Ethyl Phosphotriester (Et) Modified Rp-Rp and Sp-Sp Duplexes, {d[GGAA(Et)TTCC]}2,xe2x80x9d Nucleic Acids Res. 1986, 14, 7421-37.
(c) Pramanick, P.; Kan, L. xe2x80x9cNMR Study of the Effect of Sugar-phosphate Backbone Ethylation on the Stability and Conformation of DNA Double Helix,xe2x80x9d Biochemistry 1987, 26, 3807-12.
(d) Koole, L. H.; van Genderen, M. H. P.; Buck, H. M. xe2x80x9cA Parallel Right-Handed Duplex of the Hexamer d(TpTpTpTpTpT) with Phosphate Triester Linkages,xe2x80x9d J. Am. Chem. Soc. 1987, 109, 3916-21 *[The synthetic chemistry and hybridization data reported in this 1987 paper differed from that described later and subsequently retracted by Buck, H. M.; Moody, H. M.; Quaedflieg, P. J. L. M.; Koole, L. H.; van Genderen, M. H. P.; Smit, L.; Jurriaans, S.; Geelen, J. L. M. C.; Goudsmit, J. xe2x80x9cInhibition of HIV-1 Infectivity by Phosphate-Methylated DNA: Retraction,xe2x80x9d Science 1990, 250, 125-26 (also see: Maddox, J. xe2x80x9cDutch Cure for AIDS is Discredited,xe2x80x9d Nature 1990, 347, 411).].
(e) Quaedflieg, P. J. L. M.; Koole, L. H.; van Genderen, M. H. P.; Buck, H. M. xe2x80x9cA structural Study of Phosphate-methylated d(CpG)n and d(GpC)n DNA oligomers. Implications of Phosphate Shielding for the Isomerization of B-DNA into Z-DNA,xe2x80x9d Recl. Trav Chim Pay-Bas 1989, 108, 421-23.
(f) Quaedflieg, P. J. L. M.; Broeders, N. L. H. L.; Koole, L. H.; van Genderen, M. H. P.; Buck, H. M. xe2x80x9cConformation of the Phosphate-methylated DNA Dinucleotides d(CpC) and d(TpC). Formation of a Parallel Miniduplex Exclusively for the S-Configuration at Phosphorus,xe2x80x9d J. Org. Chem. 1990, 55, 122-27.
(g) Quaedflieg, P. J. L. M.; van der Heiden, A. P.; Koole, L. H.; Coenen, A. J. J. M.; van der Wal, S.; Meijer, E. M. xe2x80x9cSynthesis and Conformational Analysis of Phosphate-methylated RNA Dinucleotides,xe2x80x9d. J. Org. Chem. 1991, 56, 5846-59.
(15) Using modified triester phosphate oligos as probes for elucidating specific interactions with proteins:
(a) Weinfeld, M.; Drake, A. F.; Saunders, J. K.; Paterson, M. C. xe2x80x9cStereospecific Removal of Methyl Phosphotriesters from DNA by an Escherichia coliada+Extract,xe2x80x9d Nucleic Acids Res. 1985, 13, 7067-77.
(b) Gallo, K. A.; Shao, K; Phillips, L. R.; Regan, J. B.; Kozielkiewicz, M.; Uznanski, B.; Stec, W. J.; Zon, G. xe2x80x9cAlkyl Phosphotriester Modified Oligodeoxyribonucleotides. V. Synthesis and Absolute Configuration of Rp and Sp Diastereomers of an Ethyl Phosphotriester (Et) Modified EcoRI Recognition Sequence, d[GGAA(Et)TTCC]. A Synthetic Approach to Regio- and Stereospecific Ethylationinterference Studies,xe2x80x9d Nucleic Acids Res. 1986, 14, 7405-20.
(c) Koziollciewicz, M.; Stec, W. J. xe2x80x9cApplication of Phosphate-backbone-modified Oligonucleotides in the Studies on EcoRI Endonuclease Mechanism of Action,xe2x80x9d Biochemistry 1992, 31, 9460-66.
(16)
(a) Miller, P. S.; Fang, K. N.; Kondo, N. S.; Ts""O, P.O.P. xe2x80x9cSynthesis and Properties of Adenine and Thymidine Nucleoside Alkyl Phosphotriesters, the Neutral Analogs of Dinucleoside Monophosphates,xe2x80x9d J. Am. Chem. Soc. 1971, 93, 6657-65.
(b) Miller, P. S.; Barrett, J. C.; Ts""O, P.O.P. xe2x80x9cSynthesis of Oligodeoxyribo-nucleotide Ethyl Phosphotriesters and Their Specific Complex Formation with Transfer Ribonucleic Acid,xe2x80x9d Biochemistry 1974, 13, 4887-96 (and the following paper in that journal as well).
(c) Pless, R. C.; Ts""O, P.O.P. xe2x80x9cDuplex Formation of a Nonionic Oligo(deoxythymidylate) Analogue [Heptadeoxythymidylyl-(3xe2x80x2-5xe2x80x2)-deoxythymidine Heptaethyl Ester (d-[Tp(Et)17T)] with Poly(deoxyadenylate). Evaluationof the Electrostatic Interaction,xe2x80x9d Biochemistry 1977, 16, 1239-50.
(d) Miller, P. S.; Braiterman, L. T.; Ts""O, P.O.P. xe2x80x9cEffects of a Trinucleotide Ethyl Phosphotriester, Gmp(Et)Gm(Et)U, on Manmmalian Cells in Culture,xe2x80x9d Biochemistry 1977, 16, 1988-96.
(e) Petrenko, V. A.; Pozdnyakov, P. l.; Kipriyanov, S. M.; Boldyrev, A. N.; Semyonova, L. N.; Sivolobova, G. F. xe2x80x9cSite-localized Mutagenesis Directed by Phosphotriester Analogs of Oligonucleotides,xe2x80x9d Bioorg. Khim. 1986, 12, 1088-1100.
(f) Asseline, U.; Barbier, C.; Thuong, N. T. xe2x80x9cOligothymidylates Comportant La Structure Alternee Alkylphosphotriester-phosphodiester et Lies de Facon Covalente a un Agent Intercalant,xe2x80x9d Phosphorus Sulfur 1986, 26, 63-73.
(g) Marcus-Sekura, C. J.; Woerner, A. M.; Shinozuka, K.; Zon, G.; Quinnan, Jr., G. V. xe2x80x9cComparative Inhibition of Cloramphenicol Acyltransferase Gene Expression by Antisense Oligonucleotide Analogs Having Alkyl Phosphotriester, Methylphosphonate and Phosphorothioate Linkages,xe2x80x9d Nucleic Acids Res. 1987, 15, 5749-63.
(h) see ref. 6a.
(i) Koole, L. H.; van Genderen, M. H. P.; Reiniers, R. G.; Buck, H. M. xe2x80x9cEnhanced Stability of a Watson and Crick DNA Duplex Structure by Methylation of the Phosphate Groups in One Strand,xe2x80x9d Proc. K Ned. Akad. Wet. B 1987, 90, 41-6.*
(j) Petrenko, V. A.; Kipriyanov, S. M.; Boldyrev, A. N.; Pozdnyakov, P. I. xe2x80x9cMutagenesis Directed by Phosphotriester Analogues of Oligonucleotides: a Way to Site-specific Mutagenesis In Vivo,xe2x80x9d FEBS Lett. 1988, 238, 109-12.
(k) Durand, M. Maurizot, J. C.: Asseline, U.; Barbier, C.; Thuong, N. T.; Hxc3xa9lxc3xa9ne, C. xe2x80x9cOligothymidylates Covalently Linked to an Acridine Derivative and with Modified Phosphodiester Backbone; Circular Dichroism Studies of Their Interactions with Complementary Sequences,xe2x80x9d Nucleic Acids Res. 1989, 17, 1823-36.
(17) For recent reviews see:
(a) Zon, G. xe2x80x9cBrief Overview of Control of Genetic Expression by Antisense Oligonucleotides and In Vivo Applications,xe2x80x9d Mol. Neurobiology 1995, 10, 219-29.
(b) Kiely, J. S. xe2x80x9cRecent Advances in Antisense Technology,xe2x80x9d Ann. Rep. Med. Chem. 1994, 29, 297-306.
(c) Stein, C. A.; Cheng, Y.-C. xe2x80x9cAntisense Oligonucleotides as Therapeutic Agents-Is the Bullet Really Magic,xe2x80x9d Science 1993, 261, 1004-11.
(d) Varma, R. S. xe2x80x9cSynthesis of Oligonucleotide Analogues with Modified Backbones,xe2x80x9d SYNLETT 1993, 621-37.
(e) Beaucage, S. L.; Iyer, R. P. xe2x80x9cThe Functionalization of Oligonucleotides Via Phosphoramidite Derivatives,xe2x80x9d Tetrahedron 1993, 49, 1925-63.
(f) Toulmxc3xa9, J. J. in Antisense RNA and DNA; Murray, J. A. H., Ed.; Wiley, Inc.: New York, 1992, pp 175-94.
(g) IEnglisch, U.; Gauss, D. H. xe2x80x9cChemically Modified Oligonucleotides as Probes and Inhibitors,xe2x80x9d Angew. Chem. Int. Ed. EngI. 1991, 30, 613-722.
(h) Uhlmann, E.; Peyman, A. xe2x80x9cAntisense Oligonucleotides: A New Therapeutic Principle,xe2x80x9d Chem. Rev. 1990, 90, 543-84.
(i) Goodchild, J. xe2x80x9cConjugates of Oligonucleotides and Modified Oligonucleotides: A Review of Their Synthesis and Properties,xe2x80x9d Bioconjugate Chem. 1990, 1, 165-87.
(j) Hxc3xa9lxc3xa9ne, C.; Toulmxc3xa9, J.-J. xe2x80x9cSpecific Regulation of Gene Expression by Antisense, Sense, and Antigene Nucleic Acids,xe2x80x9d Biochim. Biophys. Acta 1990, 1049, 99-125.
(k) Goodchild, J. xe2x80x9cInhibition of Gene Expression by Oligonucleotides,xe2x80x9d in Oligonucleotides: Antigenge Inhibitors of Gene Expression; Cohen, J. S., Ed.; MacMillan Press; London, 1989, pp. 53-77.
(l) Zon, G. xe2x80x9cOligonucleotide Analogues as Potential Chemotherapeutic Agents,xe2x80x9d Pharm. Res. 1988, 5, 539-49.
(m) Stein, C. A.; Cohen, J. S. xe2x80x9cOligonucleotides as Inhibitors of Gene Expression: a Review,xe2x80x9d Cancer Res. 1988, 48, 2659-68.
(n) Miller, P. S.; Ts""O, P.O.P. xe2x80x9cOligonucleotide Inhibitors of Gene Expression in Living Cells: New Opportunities in Drug Design,xe2x80x9d Annu. Rep. Med. Chem. 1988, 23, 295-304.
(o) Miller, P. S.; Agris, C. H.; Blake, K. R.; Murakami, A.; Spitz, S. A.; Reddy, M. P.; Ts""O, P.O.P. xe2x80x9cNonionic Oligonucleotide Analogs as New Tools for Studies on the Structure and Function of Nucleic Acids in Living Cells,xe2x80x9d in Nucleic Acids: The Vectors of Life; Pullman, B.; Jorter, J., Eds.; D. Reidel Publishing Co.: Dordrecht, Netherlands; 1983, pp. 521-35.
(18) A review bas proposed the use of the acronym SNAIGE (Synthetic or Small Nucleic Acid Interfering with Gene Expression) as a term for describing the various approaches to genetic regulation with oligonucleotides: Leonetti, J. P.; Degols, G.; Clarenc, J. P.; Mechti, N.; Lebleu, B. xe2x80x9cCell Delivery and Mechanism of Action of Antisense Oligonucleotides,xe2x80x9d Prog. NucI. Acid Res. 1993, 44, 143-66.
(19) Zhou, Q.; Turnbull, K. D. xe2x80x9cPhosphotriesters from Tandem Phosphodiester Alkylation with Quinone Methides Followed by Lactonization,xe2x80x9d manuscript near completion for submission to: J. Am. Chem. Soc. 1997, 119.
(20) Reliable Biopharmaceuticals (St. Louis, Mo.), a supplier of oligonucleotide derivatives for antisense and antigene applications, has expressed interest in this work. We are conducting preliminary experiments to determine the potential for collaborative development of synthetic methodology for oligonucleotide modification.
(21) Sigman, D. S.; Mazunider, A.; Perrin, D. M. xe2x80x9cChemical Nucleases,xe2x80x9d Chem. Rev. 1993, 93, 2295-316.
(22) More recent examples include:
(a) Jubian, V.; Dixon, R. P.; Hamilton, A. D. xe2x80x9cMolecular Recognition and Catalysis. Acceleration of Phosphodiester Cleavage by a Simple Hydrogen-Bonding Receptor,xe2x80x9d J. Am. Chem. Soc. 1992, 114, 1120-21.
(b) Browne, K. A.; Bruice, T. C. xe2x80x9cChemistry of Phosphodiesters, DNA and Models. 2. The Hydrolysis of Bis(8-hydroxyquinoline) Phosphate in the Absence and Presence of Metal Ions,xe2x80x9d J. Amer. Chem. Soc. 1992, 114, 4951-58.
(c) Smith, J.; Ariga, K.; Anslyn, E. V. xe2x80x9cEnhanced Ilmidazole-Catalyzed RNA Cleavage Induced by a Bis-Allcylguanidimum Receptor,xe2x80x9d J. Am. Chem. Soc. 1993, 115, 362-64.
(d) Takasaki, B. K.; Chin, J. xe2x80x9cSynergistic Effect Between La(lfl) and Hydrogen Peroxide in Phosphate Diester Cleavage,xe2x80x9d J. Am. Chem. Soc. 1993, 115, 9337-38.
(e) Hall, J. Husken, D.; Pieles, U.; Moser, H. E.; Haner, R. Chemistry and Biology 1994, 1, 185-90.
(f) Bashkin, J. K.; Frolova, E. I.; Sampath, U. xe2x80x9cSequence-Specific Cleavage of HIV MRNA by a Ribozyme Mimic,xe2x80x9d J. Am. Chem. Soc. 1994, 116, 5981-82.
(g) Magda, D.; Miller, R. A.; Sessler, J. L.; Iverson, B. L. xe2x80x9cSite-Specific Hydrolysis of RNA by Europium(m) Texaphyrin Conjugated to a Synthetic Oligodeoxyribonucleotide,xe2x80x9d J. Am. Chem. Soc. 1994, 116, 7439-40.
(h) Linkletter, B.; Chin, J. xe2x80x9cRapid Hydrolysis of RNA with a CuII Complex,xe2x80x9d Angew. Chem. Int. Ed. Engl. 1995, 34, 472-74.
(23)
(a) Wilbur, D. S. xe2x80x9cRadiohalogenation of Proteins: An Overview of Radionuclides, Labeling Methods, and Reagents for Conjugate Labelling,xe2x80x9d Bioconjugate Chem. 1992, 3, 433-471.
(b) Peters, K.; Richards, F. M. xe2x80x9cChemical Cross-linking: Reagents and Problems in Studies of Membrane Structure,xe2x80x9d Ann. Rev. Biochem. 1977, 46, 523-51.
(c) Ji, T. H. xe2x80x9cThe Application of Chemical Crosslinking for Studies on Cell Membranes and the Identification of Surface Reporters,xe2x80x9d Biochim. Biophys. Acta 1979, 559, 39-69.
(24) Zhang, Y.; Chen, F.-X.; Mehta, P.; Gold, B. xe2x80x9cGroove- and Sequence-Selective Alkylation of DNA by Sulfonate Esters Tethered to Lexitropsins,xe2x80x9d Biochemistry 1993, 32, 7954-65.
(25) For reviews on quinone methides, see:
(a) Volod""kin, A. A.; Ershov, V. V. Russian Chem. Rev. 1988, 57, 336.
(b) Gruenanger, P. in Houben-Weyl Methoden der Organischen Chemie (Vol. VII/3b) Mueller, E.; Bayer, D., Eds.; G. Thieme Verlag: Stuttgart, 1979, pp. 395-521.
(c) Wagner, H.-U.; Gompper, R. in The Chemistry of Quinonoid Compounds (Vol. I) Patai, S., Ed.; John Wiley and Sons: New York, 1974, pp. 1145-1178.
(d) Turner, A. B. Quart. Rev. 1965, 18, 347.
(26) More recent, elegant examples for biomolecule alkylation include:
(a) Chatterjee, M.; Rokita, S. E. xe2x80x9cThe Role of a Quinone Methide in the Sequence Specific Alkylation of DNA,xe2x80x9d J. Am. Chem. Soc. 1994, 116, 1690-97.
(b) Li, T.; Zeng, Q.; Rokita, S. E. xe2x80x9cTarget-Promoted Alkylation of DNA,xe2x80x9d Bioconjugate Chem. 1994, 5, 497-500.
(c) Meyers, J. K.; Cohen, J. D.; Widlanski, T. S. xe2x80x9cSubstituent Effects on the Mechanism-Based Inactivation of Prostatic Acid Phosphatase,xe2x80x9d J. Am. Chem. Soc. 1995, 117, 11049-54.
(d) Myers, J. K.; Widlanski, T. S. xe2x80x9cMechanism-Based Inactivation of Prostatic Acid Phosphatase,xe2x80x9d Science 1993, 262, 1451-53.
(e) Wang, Q.; Dechert, U.; Jirik, F.; Withers, S. G. xe2x80x9csuicide Inactivation of Human Prostatic Acid Phosphatase and a Phosphotyrosine Phosphatase,xe2x80x9d Biochem. Biophys. Res. Commun. 1994, 200, 577-83.
(27) For reviews see:
(a) Sega, G. A. xe2x80x9cA Review of the Genetic Effects of Ethyl Methanesulfonate,xe2x80x9d Mutation Res. 1984, 134, 113-42.
(b) Hoffmann, G. R. xe2x80x9cGenetic Effects of Dimethyl Sulfate, Diethyl Sulfate, and Related Compounds,xe2x80x9d Mutation Res. 1980, 75, 63-129.
(c) Digenis, G. A.; Issidorides, C. H. xe2x80x9cSome Biochemical Aspects of N-Nitroso Compounds,xe2x80x9d Bioorganic Chem. 1979, 8, 97-137.
(28)
(a) Swenson, D. H.; Lawley, P. D. xe2x80x9cAlkylation of Deoxyribonucleic Acid by Carcinogens Dimethylsulfate, Ethyl Methanesulfonate, N-Ethyl-N-nitrosourea and N-Methyl-N-nitrosourea,xe2x80x9d Biochem. J. 1978, 171, 575-87.
(b) Jensen, D. E.; Reed, D. J. xe2x80x9cReaction of DNA with Alkylating Agents. Quantitation of Alkylation by Ethylnitrosourea of Oxygen and Nitrogen Sites on Poly[dA-dT] Including Phosphotriester Formation,xe2x80x9d Biochemistry 1978, 17, 5098-107.
(c) Sun, L.; Singer, B. xe2x80x9cThe Specificity of Different Classes of Ethylating Agents Towards Various Sites of HeLa DNA in vitro and in vivo,xe2x80x9d Biochemistry 1975, 14, 1795-1802.
(29) Angle, S. R.; Arnaiz, D. O.; Boyce, J. P.; Frutos, R. P.; Louie, M. S.; Mattson-Arnaiz, H. L.; Rainier, J. D.; Turnbull, K. D.; Yang, W. xe2x80x9cFormation of Carbon-Carbon Bonds via Quinone MethideInitiated Cycization Reactions,xe2x80x9d J. Org. Chem. 1994, 59, 6322-6337.
(30) Organic Synthesis, Smith, M. B.; McGraw-Hill, Inc.: New York; 1994, pp. 108-119.
(31) Schulz, W. G.; Nieman, R. A.; Skibo, E. B. xe2x80x9cEvidence for DNA Phosphate Backbone Alkylation and Cleavage by Pyrrolo[1,2-a]benzimidazoles: Small Molecules Capable of Causing Base-Pair-Specific Phosphodiester Bond Hydrolysis,xe2x80x9d Proc. NatI. Acad. Sci. USA 1995, 92, 11854-58.
(32)
(a) Tomasz, M.; Lipman, R. xe2x80x9cAlkylation Reactions of Mitomycin C at Acid pH,xe2x80x9d J. Am. Chem. Soc. 1979, 101, 6063-67.
(b) Iyengar, B. S.; Dorr, T. R.; Remers, W. A.; Kowal, C. D. xe2x80x9cNucleotide Derivatives of 2,7-Diaxninomitosene,xe2x80x9d J. Med. Chem. 1988, 31, 1579-85.
(33) Gohil, R. N.; Roth, A. C.; Day, R. A. xe2x80x9cReversible Covalent Modification of DNA,xe2x80x9d Arch. Biochem. Biophys. 1974, 165, 297-312.
(34) Bhat, G., Roth, A. C.; Day, R. A. xe2x80x9cExtrinsic Cotton Effect and Helix-Coil Transition in a DNAPolycation Complex,xe2x80x9d Biopolymers 1977, 16, 1713-24.
(35) For a thorough review, see: Hannon, C. L.; Anslyn, E. V. xe2x80x9cThe Guanidinium Group: Its Biological Role and Synthetic Analogs,xe2x80x9d Bioorg. Chem. Frontiers 1993, 3, 193-255.
(36) For additional examples, see:
(a) Blasko, A.; Dempcy, R. O.; Minyat, E. E.; Bruice, T. C. xe2x80x9cAssociation of Short-Strand DNA Oligomers with Guanidiium-Linked Nucleosides. A Kinetic and Thermodynamic Study,xe2x80x9d J. Am. Chem. Soc. 1996, 118, 7892-99.
(b) Dempcy, R. O.; Browne, K. A.; Bruice, T. C. xe2x80x9cSynthesis of the Polycation Thymidyl DNG, Its Fidelity in Binding Polyanionic DNA/RNA, and the Stability and Nature of the Hybrid Complexes,xe2x80x9d J. Am. Chem. Soc. 1995, 117, 6140.
(c) Hashimoto, H.; Nelson, M. G.; Switzer, C. xe2x80x9cFormation of Chimeric Duplexes Between Zwitteriomc and Natural DNA,xe2x80x9d J. Org. Chem. 1993, 58, 4194-95.
(d) Hashimoto, H.; Nelson, M. G.; Switzer, C. xe2x80x9cZwitterionic DNA,xe2x80x9d J. Am. Chem. Soc. 1993, LL5, 7128-34.
(e) Letsinger, R. L.; Singman, C. N.; Histand, G.; Salunkhe, M. xe2x80x9cCationic Oligonucleotides,xe2x80x9d J. Am. Chem. Soc. 1988, 115, 7128.
(1) Furberg, S.; Solbakk, J. xe2x80x9cOn the Stereochemistry of the Interaction Between Nucleic Acids and Basic Protein Side Chains,xe2x80x9d Acta Chem. Scand. B 1974, 28, 481-83.
(37) Saenger, W. Principles of Nucleic Acid Structure, Springer-Verlag: New York, 1984, pp. 385-431.
(38) Pullman, B. xe2x80x9cMolecular Mechanisms of Specificity in DNA-Antitumor Drug Interactions,xe2x80x9d in Advances in Drug Research, Testa, B., Ed.; Academic Press; London; Vol. 18, 1989, pp. 1-113.
(39)
(a) Zhou, Q.; Turnbull, K. D. xe2x80x9cEquilibrium Control in Phosphodiester Alkylation with Quinone Methides,xe2x80x9d manuscript in preparation for submission to: J. Org. Chem. 1997, 62.
(b) Zhou, Q.; Dyer, R. G.; Turnbull, K. D. xe2x80x9cA Study of Triallcylphosphate Hydrolysis Rates Related to a DNA Modifying Reagent,xe2x80x9d manuscript in preparation for submission to: J. Org. Chem. 1997, 62.
(c) Dyer, R. G.; Turnbull, K. D. xe2x80x9cPhotolytic-Initiated Formation of Quinone Methides for Phosphodiester Alkylation,xe2x80x9d manuscript in preparation.
(40) Patent Pending for xe2x80x9cBiomolecular Labelingxe2x80x9d (Apr. 4, 1997).
(41) Dyall, L. K.; Winstein, S. xe2x80x9cNuclear Magnetic Resonance Spectra and Characterization of Some Quinone Methides,xe2x80x9d J. Am. Chem. Soc. 1972, 94, 2196-99.
(42) Product identity was readily apparent from the distinct 3-bond phosphorus-hydrogen coupling constant of 8.2 Hz for the two different types of benzylic resonances in the 1H NMR spectra with an integrated ratio of 2:1. 1H NMR (CDCl3/CD3CN (1:1), 300 MHz) xcx9c7.30 (in, 10H, 2(C6HS)), 6.87 (s, 2H, C6H2), 4.95 (d, J=8.2 Hz, 4H, 2(CII2Ph)), 4.84 (d, J=8.2 Hz, 2H, CLi2Ar), 2.13 (s, 6H, 2(CH3)).
(43) For leading references, see: McCracken, P. G.; Bolton, J. L.; Thatcher, G. R. J. xe2x80x9cCovalent Modification of Proteins and Peptides by the Quinone Methide from 2-tert-Butyl-4,6-dimethylphenol: Selectivity and Reactivity with Respect to Competitive Hydration,xe2x80x9d J. Org. Chem. 1997, 62, 1820-25.
(44) Acylation of 3,5-dimethyl-4-hydroxybenzaldehyde followed by NaBH4 reduction and phosphorylation of the benzyl alcohol afforded 9 (FIG. 4).
(a) The phosphorylation reaction was a modification of: Silverberg, J. J.; Dillon, J. L.; Vemishetti, P. xe2x80x9cA Simple, Rapid and Efficient Protocol for the Selective Phosphorylation of Phenols with Dibenzylphosphite,xe2x80x9d Tetrahedron Lett. 1996, 37, 771-74.
(b) A recent paper reports on the hydrolytic stability of benzyltrialkylphosphates and the effects of various substituents on the phenyl ring: Meier, C.; Habel, L. W.; Baizarini, J.; De Clercq, E. xe2x80x9c5xe2x80x2,5xe2x80x2-Di-O-nucleosyl-Oxe2x80x2-benzylphosphotriesters as Potential Prodrugs of 3xe2x80x2-Azido-2xe2x80x2,3xe2x80x2-dideoxythymidine-5xe2x80x2-monophosphate,xe2x80x9d Liebigs Ann 1995, 2203-08.
(45) Studies of various electron-donating and electron-withdrawing substituents on the quinone methide ring and at the benzylic methylene have demonstrated their influence on quinone methide formation, reactivity and product stability:
(a) Bolton, J. L.; Comeau, E.; Vukomanovic, V. xe2x80x9cThe Influence of 4-Alkyl Substituents on the Formation and Reactivity of 2-Methoxy-Quinone Methides: Evidence That Extended xcfx80-Conjugation Stabilizes the Quinone Methide Formed From Eugenol,xe2x80x9d Chem-Biol. Interactions 1995, 95, 279-90.
(b) Thompson, D. C.; Perera, K. xe2x80x9cInhibition of Mitochondrial Respiration by a Para-Quinone Methide,xe2x80x9d Biochem. Biophys. Res. Commun. 1995, 209, 6-11.
(c) Lycka, A.; Snobl, D.; Koutek, B.; Pavlickova, L.; Soucek, M. xe2x80x9c13C NMR Study of Substituted Quinone Methides. 2- and 2,6-Substituted Fuchsones,xe2x80x9d Coll. Czech. Chem. Commun. 1981, 46, 1775-87.
(d) Velek, J.; Koutek, B.; Musil, L.; Vasickova, S.; Soucek, M. xe2x80x9cIR Spectra of Some Quinone Methides. A Study of the ortho-Effect,xe2x80x9d Coll. Czech. Chem. Commun. 1981, 46, 873-83.
(46)
(a) Turnbull, K. D. xe2x80x9cpara-Quinone.Methides: Chemistry and Exploitation as Intermediates for the Intramolecular Formation of Carbon-Carbon Bonds and Investigations into the Chemistry and Synthesis of Neolignans Via a Proposed Intermediate in Their Biosynthesis,xe2x80x9d Ph.D. Dissertation, University of California, Riverside, 1991.
(b) Angle, S. R.; Turnbull, K. D. xe2x80x9cPara-Quinone Methide Initiated Cycization Reactions,xe2x80x9d J. Am. Chem. Soc., 1989, 111, 1136. 
(47) A Fries rearrangement on 2,4-dimethylphenol followed by benzylation of the phenol, a BaeyerVilliger oxidation, and saponification of the intermediate acetate on workup afforded the required monoprotected catechol. A substitution reaction with bromoacetic acid provided the intermediate carboxylic acid which was converted to the desired etser derivatives through carbodiimide activation in the presence of the required alcohol. The dimethylamide derivative was produced directly from addition of bromoacetamide followed by methylation. Hydrogenolysis provided the various derivatives of 11 shown in the table of scheme 3 for oxidation to the desired quinone methides.
(48) Reliable Biopharmaceuticals, St. Loius, Mo., has generously donated multi-milligram quantities of TpT (with and without protecting groups) and CpA (with and without protecting groups) for the purpose of these studies.
(49) Wakselman, M. xe2x80x9c1,4- and 1,6-Eliminations from Hydroxy- and Amino-substituted Benzyl Systems: Chemical and Biochemical Applications,xe2x80x9d Nouv. J. Chim. 1983, 7, 439-47.
(50)
(a) Kanamathareddy, S.; Gutsche, C. D. xe2x80x9cCalixarenes: Selective Functionalization and Bridge Building,xe2x80x9d J. Org. Chem. 1995, 60, 6070-75.
(b) Adam, I.; Sharma, S. K.; Gutsche, C. D. xe2x80x9cThe Quinonemethide Route to Mono- and Tetrasubstituted Calix[4]arenes,xe2x80x9d J. Org. Chem. 1994, 59, 3716-20.
(c) Note that even aniline has been eliminated to produce quinone methides: Angle, S. R.; Yang, W. xe2x80x9cSynthesis and Chemistry of a Quinone Methide Model for Anthracycline Antitumor Antibiotics,xe2x80x9d J. Am. Chem. Soc. 1990, 112, 4524-28.
(51) Quinone methides have been generated by laser flash photolysis (266 nm) of phenolic benzyl alcohols and the UV of the transient intermediate was presumed to be the quinone methide. These are differentiated from caged quinone methide precursors which can be irradiated at wavelengths outside 350 nm in order to be useful in the presence of biological molecules: Diao, L.; Yang, C.; Wan, P. xe2x80x9cQuinone Methide Intermediates from the Photolysis of Hydroxybenzyl Alcohols in Aqueous Solution,xe2x80x9d J. Am. Chem. Soc. 1995, 117, 5369-70.
(52) This allows transmission of  greater than 350 nm wavelength which will prevent absorbance of biological aromatics.
(53) Fleming, S. A.; Jensen, A. W. xe2x80x9cSubstituent Effects on the Photocleavage of Beazyl-Sulfur Bonds. Observation of the Meta-Effectxe2x80x9d J. Org. Chem. 1996, 61, 7040-44, and references therein.
(54)
(a) Dimethoxybenzoin: Pirrung, M. C. Shuey, S. W. xe2x80x9cPhotoremovable Protecting Groups for Phosphorylation of Chiral Alcohols. Asymmetric Synthesis of Phosphotriesters of (xe2x88x92)-3xe2x80x2,5xe2x80x2-Dimethoxybenzoin,xe2x80x9d J. Org. Chem. 1994, 59, 3890-97.
(b) Sheehan, J. C.; Wilson, R. M.; Oxford, A. W. xe2x80x9cThe Photolysis of Methoxy-Substituted Benzoin Esters. A Photosensitive Protecting Group for Carboxylic Acids,xe2x80x9d J. Am. Chem. Soc. 1971, 93, 7222-28.
(c) a-Methyl-4,5-dimethoxy-2-nitrobenzyl: Marriott, G. xe2x80x9cCaged Protein Conjugates and Light-Directed Generation of Protein Activity: Preparation, Photoactivation, and Spectroscopic Characterization of Caged G-Actin Conjugates,xe2x80x9d Biochemistry 1994, 33, 9092-97.
(d) a-Carboxy-2-nitrobenzyl: Gee, K. R.; Wieboldt, R.; Hess, G. P. xe2x80x9cSynthesis and Photochemistry of a New Photolabile Derivative of GABA. Neurotransmitter Release and Receptor Inactivation in the Microsecond Time Region,xe2x80x9d J. Am. Chem. Soc. 1994, 116, 8366-67.
(55) For reviews of photolabile protecting groups, see:
(a) Adams, S. R.; Tsien, R. Y. xe2x80x9cControlling Cell Chemistry with Caged Compounds,xe2x80x9d Annu. Rev. Physiol. 1993, 55, 755-84.
(b) Gurney, A. M.; Lester, H. A. xe2x80x9cLight Flash Physiology with Synthetic Photosensitive Compounds,xe2x80x9d Physiol. Rev. 1987, 67, 583-615.
(c) Pillai, V. N. R. xe2x80x9cPhotoremovable Protecting Groups in Organic Synthesis,xe2x80x9d Synthesis 1980, 1-26.
(56)
(a) Derivatives with dimethyl substituents and a benzylic methyl (R, R1, R11xe2x95x90Me) were prepared by acylation of 2,6-dimethylphenol followed by a Fries rearrangement to the 4-methylketone derivative followed by protection with o-nitrobenzyl bromide and NaBH4 reduction to the benzyl alcohol derivative. The benzyl alcohol was converted to the acetate, trifluoromethyl acetate, bromide or chloride through literature methods.
(b) Derivatives with no substituents (R, R1, R11=H) were made by protection of 4-hydroxybenzaldehyde with o-nitrobenzyl bromide and reductive amination with the desired dialkylanuine.
(c) The methoxy substituted derivative (R, R11=H, R=OMe) were made by protecting vanillin with o-nitrobenzyl bromide, NaBH4 reduction and conversion to the chloride with N-chlorosuccinimide.
(d) Dimethyl substituted derivatives with no benzylic alkyl group (R, R1=Me, R11=H) were prepared from protection of 3,5-dimethyl-4-hydroxybenzaldehyde with the alcohol of the desired photolabile protecting group in the presence of triphosgene to afford the carbonate protected benzaldehyde. NaBH4 reduction and conversion to the acetate or chloride afforded the desired products.
(57) Besides the experiment reported (FIG. S5), we have run several experiments with guanylated octopamine (structure at right) trying to induce quinone methide formation via dehydration. This has been attempted in refluxing TFA with no sign of any type of reaction. We have also obtained preliminary results on the benzylic chlorination of this compound in DMF with phosphoryl chloride. NMR evidence suggests quantitative conversion to the benzyl chloride with no cyclization of the guanidine. Treatment of this intermediate with NaOH resulted in apparent B-elimination to the styrene, but no sign of cyclization was evident by 1H NMR.
(58) Athough the possibility for this cyclization is readily apparent, a thorough examination of the natural product, synthetic, and medicinal literature has thus far failed to turn up an example of a cyclization such as could occur in any of our proposed ATAR systems (a 3 to 7-exo-tet or trig depending on which system and whether the quinone methide precursor or the quinone methide is cyclizing (see A, B, or C in section 4.3)). Although there are limited examples of guanidine SN2 or conjugate addition reactions, these require deprotonation under strong basic conditions or electrophilic activation under acidic conditions [For a recent example of an SN2 reaction see: Vaidyanathan, G.; Zalutsky, M. R. xe2x80x9cA New Route to Guanidines from Bromoalkanes,xe2x80x9d J. Org. Chem. 1997, 62, 4867-69.]. Guanidine condensation reactions are well precedented and a useful means for making heterocyclic structures and in natural product synthesis and generally require acid or base catalysis to drive the reactions [For leading references, see:
(a) ref 35.
(b) Yamamoto, Y.; Kojima, S. xe2x80x9cSynthesis and Chemistry of Guanidine Derivatives,xe2x80x9d in The Chemistry of Amidines and Amidates, Patai, S.; Rappoport, Z., Eds.; John Wiley and Sons: N.Y. 1991, 485-526.
(c) Berlinck, R. G. S. xe2x80x9cNatural Guanidine Derivatives,xe2x80x9d Nat. Prod. Rep. 1996, 377-409.].
(59) One example of a natural product which has been isolated which has the potential to undergo a 5-exo-trig cyclization was found. This is martinelline: (Witherup, K. M.; Ransom, R. W.; Graham, A. C.; Bernard, A. M.; Salvatore, M. J.; Lumma, W. C.; Anderson, P. S.; Pitzenberger, S. M.; Varga, S. L. xe2x80x9cMartinelline and Martinellic Acid, Novel G-Protein linked Receptor Antagonist from the Tropical Plant Martinella iquitosensis (Bignoniaceae),xe2x80x9d J. Am. Chem. Soc. 1995, 117, 6682-85.). Although this was not specifically sought out, the natural product was isolated as the 3xc3x97TFA salt and stable to the extraction, characterization and assaying processes. A small amount of the correponding acid which would result from cleavage of the ester bond was also present; but the authors were uncertain if it was an artifact or natural. It was reported as a natural product.
(60) This is definitively stated as all the components produced in the reaction mixture have been independently characterized by 1H NMR, and there is no visible sign of any new benzylic resonances, or ethyl resonances which would identify the addition of the ethyl guanidine to the quinone methide.
(61) It is believed that cyclization will not be a concern in the ATAR as long as the guanidine is protonated. Guanidine deprotection will be the final step in our synthetic protocol and will result in the protonated guanidinium ATAR which will remain under aqueous conditions for all uses.
(62) Bernatowicz, M. S.; Wu, Y.; Matsueda, G. R. xe2x80x9c1H-Pyrrazole-1-carboxamidine Hydrochloride: An Attractive Reagent for Guanylation of Arnines and Its Application to Peptide Synthesis,xe2x80x9d J. Org. Chem. 1992, 57, 2497-502.
(63) lodination of phthalimide-protected octopamine followed by a Stille reaction with tetramethyltin has been accomplished in approx. 98% yield for the two steps.
(64) Three approaches are underway in order to establish different chemistry which will be utilized for more advanced derivatives:
(a) Graduate student Tony Hudgens has accomplished the diiodination of octopamine followed by Stille methylation, guanylation, and protection of the phenol as the silyl ether. He is presently optimizing a benzyl alcohol reduction and deprotection sequence to afford 20 (Rxe2x80x2xe2x80x3=H). These steps have already been accomplished in related systems.
(b) Another approach, nearly complete, is being carried out by Mr. Damon Arinitage, a senior Honor""s student in our laboratories. This has involved a condensation of nitromethane with benzyl-protected 3,5-dimethyl-4-hydroxy benzaldehyde followed by dehydration according to a literature procedure (Wollenberg, R. H.; Miller, S. J. xe2x80x9cNitroalkane Synthesis. A Convenient Method for Aldehyde Reductive Nitromethylation,xe2x80x9d Tetehedron Lett. 1978, 35, 3219-22.). Hydrogenation accomplishes the debenzylation, stryrene reduction, and reduction of the nitro to the amine. The amine is being guanylated using standard procedures to afford 20 (Rxe2x80x3xe2x80x2=H).
(c) An approach to 21 being carried out by graduate student Tony Hudgens is nearly identical to the approach described above (64a to make 20), except the phenol is protected as the o-nitrobenzyl carbonate and the benzyl alcohol is converted to the benzyl chloride by procedures already established in the group.
(65) Yamamoto, Y.; Kojima, S xe2x80x9cSynthesis and Chemistry of Guanidine Derivatives,xe2x80x9d in The Chemistry of Amidines and Amidates, Patai, S.; Rappoport, Z., Eds.; John Wiley and Sons: N.Y. 1991, 485-526.
(66) Cliffe, l. A. xe2x80x9cFunctions Containing an Iminocarbonyl Group and any Element Other Than a Halogen or a Chalcogen,xe2x80x9d in Comprehensive Organic Functional Group Transformations, Gilchrist, T. L., Ed.; Pergamon: U.K. 1995, 639-75.
(67) The potential for a phenyl substituent to increase intercalation is realized. As the phenyl group itself is not very polar in nature, this should not be a favored interaction.
(68) The o-nitrobenzyl carbonate protection of the phenol has proven more effective at stablizing the benzyl chloride towards hydrolysis in the final product. The Boc protecting groups are difficult to remove in high yield, so bringing the amine through as the phthaloyl-protected derivative with deprotection after the Stilie methylation followed by guanylation and then chlorination as shown has proven the preferred route in other systems (see footnote 64c and 64a).
(69)
(a) Wang, B.; Liu, S.; Borchardt, R. T. xe2x80x9cDevelopment of a Novel Redox-Sensitive Protecting Group for Amines Which Utilizes a Facilitated Lactonization Reaction,xe2x80x9d J. Org. Chem. 1995, 60, 539-43.
(b) Cauwberghs, S.; De Clercq, P. J.; Tinant, B.; Declercq, J. P. xe2x80x9cFactors Affecting Ease of Ring Formation. The Effect of Anchoring Substition on the Rate of an Intramolecular Diels-Alder Reaction with Furan-Diene,xe2x80x9d Tetrahedron Lett. 1988, 29, 2493-96.
(70) Molecular modeling (MM2) minimizations on these systems suggest there will be no steric or conformational constraints to addition of the phosphodiester to the quinone methide from the favored conformations. The highly flexible directionality and tautomerizing nature of hydrogen bonding make modeling of the transition state of little predictive value.
(71) Although we have been using more traditional brominations, we will try a new procedure which is reported to give 5% or less of the dibromo-product with phenols. Mashraqui, S. H.; Mudaliar, C. D.; Hariharasubrah-manian, H. xe2x80x9c4,4-Dimethyl-3-methylpyrazol-5-one: New Applications for Selective Monobromination of Phenols and Oxidation of Sulfides to Sulfoxides,xe2x80x9d Tettrahedron Lett. 1997, 38, 4865-68.
(72) Barluenga, J.; Garcia-Martin, M. A.; Gonzalez, J. M.; Clapes, P.; Valencia, G. xe2x80x9clodination of Aromatic Residues in Peptides by Reaction with JPy2BF4,xe2x80x9d J. Chem. Soc. Chem. Commun. 1996, 1505-06.
(73) Scott, W. J. xe2x80x9cThe Stille Reaction,xe2x80x9d Org. React. 1997, 50, 1-652.
(74) Pappas, J. J.; Keaveney, W. P.; Gancher, E.; Berger, M. xe2x80x9cA New and Convenient Method for Converting Olefins to Aldehydes,xe2x80x9d Tetrahedron Lett. 1966, 4273-78.
(75) Borch, R. F.; Bernstein, M. D.; Durst, H. D. xe2x80x9cThe Cyanohydridoborate Anion as a Selective Reducing Agent,xe2x80x9d J. Am. Chem. Soc. 1971, 93, 2897-904.
(76) De Maijere, A.; Meyer, F. E. xe2x80x9cFine Feathers Make Fine Birds: The Heck Reaction In Modern Garb,xe2x80x9d Angew. Chem. Int. Ed. Eng. 1994, 33, 2379-411.
(77) Pasto, D. J.; Taylor, R. T. xe2x80x9cReduction with Diimide,xe2x80x9d Org. React. 1991, 40, 91-155.
(78) Danishefsky, S. xe2x80x9cSiloxy Dienes in Total Synthesis,xe2x80x9d Acc. Chem. Res. 1981, 14, 400-406.
(79) Sibi, M. P.; Stessman, C. C.; Schultz, J. A.; Christensen, J. W.; Lu, J.; Marvin, M. xe2x80x9cA Convenient Synthesis of N-Methoxy-N-methylamides From Carboxylic Acids,xe2x80x9d Synth. Commun. 1995, 25, 1255-64.
(80) Thompson, C. M.; Green, D. L. C. xe2x80x9cRecent Advances in Dianion Chemistry,xe2x80x9d Tetrahedron, 1991, 47, 4223-85.
(81) Sibi, M. P. xe2x80x9cApplications of N-Methoxy-N-methylamides in Synthesis,xe2x80x9d Org. Prep. Proced. Int. 1993, 23, 15.
(82) Danishefsky, S.; Yan, C.-F.; Singh, R. K.; Gammill, R. B.; McCurry, P. M.; Fritsch, N.; Clardy, J. xe2x80x9cDerivatives of 1-Methoxy-3-trimethylsilyloxy-1,3-butadiene for Diels-Alder Reactions,xe2x80x9d J. Am. Chem. Soc. 1979, 101, 7001-008.
(83) Danishefsky has examined the cycloaddition reactions of sterically bulky dienes similar to 28 with methyl substituents at the 2- and 4-positions 82. Despite the increased sterics, reaction through the s-cis conformation is still a facile process. The same researchers demonstrated the use of dimethyl allene-1,3-dicarboxylate (29) as a dienophile in reactions with Danishefsky""s diene to afford high regioselectivity and yield of a benzene derivative with a substitution pattern similar to our target 82.
(84) Euranto, E. K. in The Chemistry of Carboxylic Acids and Esters, Patal, S., Ed.; Interscience Publishers: N.Y. 1969, pp. 505-588.
(85) This will be similar to Overman""s tandem Curtius-carbamylation reaction: Overman, L. E.; Taylor, G. F.; Petty, C. B.; Jessup, P. J. xe2x80x9ctrans-1-Nxcx9cAcylamino-1,3-dienes: Preparation From Dienoic Acid,xe2x80x9d J. Org. Chem. 1978, 43, 2164-67.
(86)
(a) Sulfonamides are the most stable amine protecting group; however, unlike most sulfonamides, this derivative is readily deprotected with fluoride ion. This will be particularly imperative as it will be the final protecting group removed in the synthesis of the fully functionalized ATAR. Weinreb, S. M.; Demko, D. M.; Lessen, T. A. xe2x80x9cB-trimethylsilylethanesulfonyl Chloride (SES-CI): A new Reagent for Protection of Amines,xe2x80x9d Tetrahedron Lett 1986, 27, 2099-2102.
(b) Alternatively, a more acid labile sulfonamide protecting group (Pmc) will be used: Ramage, R., Green, J., Blake, A. J. xe2x80x9cAn Acid Labile Arginine Derivative for Peptide Synthesis: NGxcx9c2,2,5,7,8xcx9cPentamethylchroman-6-sulfonyl-L-arginine,xe2x80x9d Tetrahedron 1991, 47, 6353-70.
(87) Hagihara, M.; Schreiber, S. L. xe2x80x9cReassignment of Stereochemistry and Total Synthesis of Thrombin Inhibitor Cyclotheonamide B,xe2x80x9d J. Am. Chem. Soc. 1992, 114, 6570-71.
(88) Chem. Pharm. Bull. 1984, 33, 1016.
(89) Sharp, M. J.; Cheng, W.; Snieckus, V. xe2x80x9cSynthetic Connections to the Aromatic Directed Metallation Reaction. Functionalized Aryl Boronic Acids by Ipso Borodesilylation. General Syntheses of Unsymmetrical Biphenyls and m-Terphenyls,xe2x80x9d Tetrahedron Lett. 1987, 28, 5093-96.
(90) A Heck approach as previously described will be used if the amine-containing functionality proves unnecessary.
(91) The guanidine will be deprotected on a derivative having an ester for lactonization.
(92) The products of these reactions will be useful in all future studies for correlation with ATAR alkylated products which can be digested down to the enzymatically protected trialkylphosphate dinucleotide components.
(93) If necessary, product analysis will take advantage of procedures used for purifying hydrophobic methylphosphonate modified oligonucleotides: Lin, S.-B.; Chang, G.-W.; Teh, G.-W. Lin, K.-I.; Au, L. C. xe2x80x9cA Simple and Rapid Method for Purification of Oligodeoxyribonucleoside Methylphosphonates,xe2x80x9d Biotechniques 1993, 14, 795-98.
(94) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning 2nd Ed. (Cold Springs Harbor Laboratory, Cold Springs Harbor, N.Y., 1989).
(95) The higher the degree of alkylation, the slower the oligo should migrate; or if the polarity is reversed, the faster they will migrate. This will be quantified by densitometry of the autoradiography to allow determination of the degree of alkylation.
(96) Alternatively, HPLC will also allow resolution and quantification of the relative degree of alkylation.
(97) i.e., Is there a difference in degree of alkylation at the ends or the middle of the oligonucleotide?
(98) Diastereomers afford differing 31P resonance signals. An NMR nano-probe would greatly facilitate these analyses, and funding for such an upgrade will be sought.
(a) Lxc3x6schner, T.; Engels, J, W. xe2x80x9cDiastereomeric Dinucleoside-methylphosphonates: Determination of Configuration with the 2-D NMR ROESY Technique,xe2x80x9d Nucleic Acids Res. 1990, 18, 5083-88.
(b) Summers, M. F.; Powell, C.; Egan, W.; Byrd, R. A.; Wilson, W. D.; Zon, G. xe2x80x9cAlkyl Phosphotriester Modified Oligodeoxyribonucleotides. VI. NMR and UV Spectroscopic Studies of Ethyl Phosphotriester (Et) Modified Rp-Rp and Sp-Sp Duplexes, {d[GGAA(Et)TTCC]}2,xe2x80x9d Nucleic Acids Res. 1986, 14, 7421-37.
(c) Pramanik, P.; Kan, L. xe2x80x9cNMR Study of the Effect of Sugar-phosphate Backbone Ethylation on the Stability and Conformation of DNA Double Helix,xe2x80x9d Biochemistry 1987, 26, 3807-12.
(99) Oligonucleotides will be synthesized on an automated synthesizer and purified according to standard protocols:
(a) Beaucage, S. L.; Caruthers, M. H. Tetrahedron Lett. 1981, 22, 1859.
(b) Sinha, N. D.; Biernat, J.; McManus, J.; Koster, H. Nucleic Acids Res. 1984, 12, 4539-57.
(100) The commercially available 1,12-dodecanediol will be protected as the 4,4xe2x80x2-dimethoxytrityl ether (Khorana, H. G. Pure Appl. Chem. 1968, 17, 349.) and converted to the synthesizer-ready phosphoramidite according to standard protocol (Gait, M. J., Ed., Oligonucleotide Synthesis, A Practical Approach, IRL Press: New York; 1990, pp. 41-45.).
(101) Based on modeling estimates, an initial linker will have 16 atoms from the 5xe2x80x2-phosphate oxygen of the oligonucleotide to the benzyl ring of the reagent.
(102) Oakley, M. G.; Turnbull, K. D.; Dervan, P. B. xe2x80x9cSynthesis of a Hybrid Protein Containing the Iron-Binding Ligand of Bleomycin and the DNA-Binding Domain of Hin,xe2x80x9d Bioconjugate Chem. 1994, 5, 242-47.
(103) Bergeron, R. J.; McManis, J. J. J. Org. Chem. 1988, 53, 3108. This can be accomplished without hydrolyzing a methyl ester, so the oligonucleQtide attachment will remain unharmed.
(104) Telser, J.; Cruickshank, K. A.; Morrison, L. E.; Netzel, T. L. xe2x80x9cSynthesis and Characterization of DNA Oligomers and Duplexes Containing Covalently Attached Molecular Labels: Comparison of Biotin, Fluorescein, and Pyrene Labels by Thermodynamic and Optical Spectroscopic Measurements,xe2x80x9d J. Am. Chem. Soc. 1989, 111, 6966-76.
(105) Han, H.; Dervan, P. B. xe2x80x9cVisualization of RNA Tertiary Structure by RNA-EDTA.Fe(II) Autocleavage: Analysis of tRNAPhe with Uridine-EDTA.Fe(II) at Position 47,xe2x80x9d Proc. NatI. Acad. Sci. USA 1994, 91, 4955-59
(106) Telser, J.; Cruickshank, K. A.; Schanze, K. S.; Netzel, T. L. xe2x80x9cDNA Oligomers and Duplexes Containing a Covalently Attached Derivative of Tris(2,2xe2x80x2-bipyridine)ruthenium(II): Synthesis and Characterization by Thermodynamic and Optical Spectroscopic Measurements,xe2x80x9d J. Am. Chem. Soc. 1989, 111, 7221-26.
(107) The standard deprotection methods will be tested in model systems first (ammonium hydroxide), and if necessary, more mild deprotection methods have been developed for solid-support oligonucleotide deprotection and cleavage of alkali-labile oligonucleotides such as with 5% K2C03 in MeOH. 12c A more labile oxallyl-CPG solid-support linkage can be used with this deprotection methodology. 12d Alternatively, in conjunction with the oxallyl-CPG, an allyloxy protection scheme can be used throughout the oligonucleotide for mild catalytic palladium deprotection, 12b which the carbonate and ester will be stable towards.
(108) A post-synthetic methodology could also be used to functionalize the deprotected and cleaved, modified oligonucleotide in solution.
(109) The absorbance ratio at 260 nm (for DNA) versus 350 nm (for the reagent).
(110) The protocol for triple helix formation and analysis by affmity cleavage is very well developed, see:
(a) Greenberg, W. A.; Dervan, P. B. xe2x80x9cEnergetics of Formation of Sixteen Triple Helical Complexes Which Vary at a Single Position Within a Purine Motif,xe2x80x9d J. Am. Chem. Soc. 1995, 117, 5016-22.
(111) Kennard, O.; Hunter, W. N. xe2x80x9cSingle-Crystal X-Ray Diffraction Studies of Oligonucleotides and Oligonucleotide-Drug Complexes,xe2x80x9d Angew. Chem. Int. Ed. Engl. 1991, 30, 1254-77.
A. Research Plan
1. Introduction
Rapid progress in sequencing the human genome1A opens new doors for potential technological developments for studying and treating disease at the foundational genetic level. One area of such potential development is the in vivo chemical modification of genomic DNA for diagnostics, therapeutics, and the study of biological processes. This requires progress in several areas of biomedical technology. Advances in oligonucleotide delivery to cells2A and sequence-specific recognition of DNA3A are two key areas. Our research program is targeting an unexplored area for the development of an innovative, chemical means to covalently deliver a variety of reporter groups, drug agents, or proteins to DNA. The ability to site-specifically attach such moieties to DNA would allow various genetic-based, biological studies to be conducted4A and provide a new means for efficient diagnostics,5A therapeutics6A and biological control at the genetic level.7A 
Covalent modification of the phosphodiester group would be of most interest as it is the common, repeating, nucleophilic functional group throughout nucleic acid polymers. Whereas covalent modification of the nucleic acid bases generally leads to strand cleavage through depurination and will disrupt base pairing by interfering with hydrogen bonding, modification of the phosphate will have less effect on nucleic acid structure and function (FIG. 1A).
This proposal will present the foundational research necessary for development of such a chemical reagent to accomplish this overall goal. This research is developing a variety of useful chemistry, synthetic methodology, and technologically applicable compounds in pursuit of the long-termn goal.
This de vivo designed reagent will covalently transfer attached molecules to a target phosphate group of a nucleic acid polymer (FIG. 1A). The reagent is designed around a quinone methide with its bimodal electrophilic and a nucleophilic reactivity. The design features include:
An independently tethered delivering oligonucleotide and molecule to be transferred (1, oligo and R, respectively, FIG. 1A). These are appended to a DNA synthesizer, machine-ready core reagent using standard automated, solid-phase, synthetic protocol for efficiency and versatility.
Phosphate specificity through a guanidinium-phosphate complex (2). The guanidinium group will be substituted as needed to lower its nucleophilicity and prevent intramolecular reaction.
xe2x80x9cCagedxe2x80x9d reactivity initiated by photolysis to afford the quinone methide precursor (3).
An intramolecular tertiary amine may be incorporated if it proves beneficial to assist 1,6-elimination8 to afford the intermediate quinone methide (4).
Alkylation of the phosphodiester with the quinone methide resulting in the in situ release of the delivering oligonucleotide through lactonization to accomplish the transfer step (4 to 5). The oligonucleotide tether is designed to be cleaved at a slower rate than 1,6-elimination occurs. The intramolecular conjugate acid will afford stability to the trialkylphosphate prior to lactonization.
Formation of a covalently stable trialkylphosphate upon lactone formation by trapping the alkylated product (5) and preventing reaction reversibility.
The reagent is an affinity transfer alkylating reagent (ATAR) for labeling the phosphodiester of nucleic acids. The research program is designed to streamline development of the ATAR by optimizing the chemistry through independent model system studies. The final reagent will be suitable for general use by attaching any delivering oligonucleotide on an automated synthesizer followed by attachment of a desired reporter group,9A drug agent,10A or protein conjugate11A on the solid support or post-synthetically. This provides a xe2x80x9cuser-friendlyxe2x80x9d reagent for use in modifying DNA, studying various nucleic acid-protein interactions, and for drug delivery applications.
The chemistry required for the ATAR is being developed through a variety of small molecule model studies. Each study requires minimal synthesis in order to independently investigate the various chemical aspects of the ATAR for optimization. Progressively more complex studies are underway in order to coordinate the compatibility of the chemical reactions for optimal control of all aspects of the ATAR design. The chemistry necessary for the total syntheses of fully functionalized derivatives for incorporation into the ATAR on a DNA synthesizer is being developed in the course of these model studies. This proposal maps out the investigations for development of the chemistry of the ATAR focusing on the model system syntheses and studies. Although the overall goal of this research is the envisioned applications of the ATAR mentioned above,4A-7A this will be beyond the time frame for which present funding is sought.
Several significant subset developments result from the pursuit of the overall research goal. One will be a simplified synthetic method12A,13A for site selective alkylation and peralkylation of oligonucleotide phosphodiesters to produce trialkylphosphate modified oligonucleotides for various uses.14A,15A,5A This backbone modification affords enhanced hybridization properties16A and antisense/antigene applications.17A,18A This research has already afforded a useful synthetic method for modifying phosphodiesters with the formation, isolation and fall characterization of trialkylphosphates.19A Some aspects of commercial potential for this methodology are being pursued with industrial support.20A A non-specific chemical nuclease is being developed in conjunction with this research and a method for the site-selective hydrolytic cleavage of DNA.21A,22A 
2. Background and Significance
Heterobifunctional crosslinking reagents containing a cleavable linker have been developed for studying protein-protein interactions;23A These reagents require an invasive chemical step to transfer the probe molecule from the delivering species to the target protein. This limits their use to in vitro applications. The ATAR we are developing will involve an in vitro cleavage step of the initially formed crosslinked complex in order to release the delivering oligonucleotide. This forms a DNA target which has been covalently modified with a small molecule carrying the independently attached label. The independent synthetic attachment of both the delivering oligonucleotide and the desired reporter group, drug agent, or protein provides a versatile ATAR for various applications.
One example of a reagent which transfers a methyl group from a nucleic acid binding reagent to a nucleic acid base in vitro comes from the work of Gold and coworkers.24A They produced a methylating reagent by tethering methyl sulfonates to a dipeptide lexitropsin, an A/T-rich minor groove binder. This reagent allowed methylation ot the adenin-N3 selectively, resulting in the iclease ot the lexitropsin sulfonic acid byproduct. The goal of this proposal is to define a method to extend this type of in vitro transfer chemistry beyond methylation to the transfer of a large variety of reporter groups or drug molecules. This will be accomplished by having them tethered to a ieactive quinorie methide25A,26A which will initiate nucleophilic attack followed by the in situ release of the delivering molecule. Further, the ATAR being developed will be latently reactive upon photolysis after binding to the target site in order to minimize secondary alkylation reactions. It will also target the phosphate residue of nucleic acids in order to mimnnze perturbalion of the bases, leaving the nucleic acid free for hybridization.
Although the phosphate residue of nucleic acids is not the chemoselective site for alkylation by many routinely used electrophilic reagents,27A in situ alkylation of the phosphodiester to afford phosphotnesters is observed. Ethylnitrosourea (ENU) shows the highest selectivity for phosphotriester formation relative to methylnitrosourea (MNU), dimethylsulfate (DMS) and ethylmerhanesultonate28A Expressed in terms of total DNA alkylation, the extent of phosphodiester alkylation by ENU has been estimated to be between 59%28A and 7Q%.28A 
A quinone methide is an effective alkylating agent with a dialkylphosphate (see Preliminary Results). A quinone methide (FIG. 2A) is a potent electrophile due to its highly polarized nature. Rearomatization of the quinone methide ring is a strong driving force for reaction.29A This relatively hard electrophile is a good alkylating agent for the hard phosphate oxygen.30A 
Skibo and coworkers have recently developed a molecule (5, FIG. 3A) that alkylates the phosphate residue of nucleic acids.31A This molecule contains a binding region which recognizes the adenine.thymine (A.T) base pair (and to a lesser extent the guanine.cytosine (G.C) base pair). The alkylating region is composed of an aziridinium moiety for selective phosphate alkylation (6, FIG. 3A) instead of normal alkylation of N-7.32A 
Day and coworkers attempted to develop a reagent for alkylation of DNA phosphate group using para-bromomethylbenzoyl choline iodide.31A Unfortunately, it was later reported the reagent was polyinerizing and phosphate alkylation was not occurring.34A This work suggests the challenge in finding a strong enough electrophile to selectively react with a phosphate. As indicated, we have already shown that quinone methides alkylate dialkylphosphates in an aqueous environment.
The guanidinium functional group is extensively used in biological systems and various artificial receptors for phosphate recognition and binding.35A This type of ionic association of cationic amine residues with DNA has been successfully used by other researchers in order to enhance binding to DNA.36A As charge-charge attractions are the strongest noncovalent molecular interactions, salt bridges between nucleic acid phosphates and positively charged amino acid side chains are individually the highest strength interactions in protein-nucleic acid interactions.17A 
The ATAR we are developing takes advantage of this type of guanidinium-phosphate ionic association to direct the alkylation process. The precursor to the quinone methide will incorporate a guanidinium residue to enhance the effective concentration of the phosphodiester. The guanidinium group may associate with other nucleic acid sites, such as the bases;35A however, the thermodynamic preference for two point hydrogen bonding and charge pairing of a guanidinium-phosphate complex is well accepted.35A,37A 
3. Preliminary Results
Various model system studies are being conducted to develop the chemistry necessary for the ATAR. Below are nine key results which contribute to the ATAR development. In the area of de novo design, the importance of a compound comes only with proven function. This often delays publication of foundational work until the significance of the chemistry is verified. The formation of isolated, fully characterized trialkylphosphates has been accomplished to provide a useful synthetic approach for modifying phosphodiesters. Due to this recent demonstration of function, publication of these results are in progress,19A and publication of the foundational work which supported it will follow.39A There is presently a provisional patent covering many of these developments.40A Some aspects of commercial potential of the phosphotriester forming reactions are being pursued with industrial support.20A 
(1) Quinone methide alkylation of a phosphodiester to form a phosphotriester. Studies of a quinone methide with a dialkylphosphate have been conducted 2,4,6-Trimethylphenol was quantitatively converted to quinone methide 7 with Ag2O41A and dibenzyl-phosphoric acid was added to produce phosphotriester 8 as the exclusive product (FIG. S2A).42A 
(2) Phosphotriester product is favored upon protonation. Formation of 8 is an equilibrium process. Trialkylphosphate 8 is favored under acidic conditions which protonate the quinone methide oxygen leading to the phenol. However, under basic conditions where the phenol is deprotonated or conditions acidic enough to protonate the phosphotriester oxygen, 7 is favored. As initially seen by the effect of various acids in the pKa range shown in FIG. T1A, this should favor phosphotriester formation under biologically relevant conditions near pH 7.
(3) Kinetic favorability of phosphotriester formation over hydrolysis in the presence of water. The reaction of quinone methide 7 and two equivalents of dibenzylphosphoric acid in the presence of excess water (xcx9c200 equivalents for a homogeneous solution) afforded only trialkylphosphate 8 as the product in the equilibrium by 1H NMR analysis. Minor amounts of the benzyl alcohol hydrolysis product was evident by 1H NMR analysis after 18 hours ai ambient temperature. Trialkylphosphate 8 is the kinetic product. A similar amount of 8 is produced in the presence of a much higher concentration of water (3,000 equivalents forming a bilayer) at ambient temperature after 30 minutes. However, the benzyl alcohol hydration product begins to drain off the kinetically formed 8 affording complete conversion to benzyl alcohol after 18 hours. Hydrolysis to benzyl alcohol appears to be the thermodynamic product. Similar results of quinone methides reacting with amino acid derivatives under aqueous conditions have been reported by other researchers.43A 
(4) Hydrolytic stability of an acetylated trialkylphosphate derivative. The effect of protonation on the alkylation reaction above and that the trialkylphosphate is the kinetic product suggested trapping of the phosphate alkylated DNA as lactone derivative 4 (FIG. S1A) should be favored over hydrolysis (to afford a benzyl alcohol) under physiological conditions. Investigation of the stability of lactone trapped trialkylphosphate product was the next step. The high stability of independently synthesized 944 (FIG. 4A) in water (pH 6.5, 40xc2x0 C., overnight) demonstrates the expected stability of the lactone product 4 (FIG. S1A) which will result from the ATAR phosphate alkylation reaction.
(5) Trapping stable phosphotriesters through tandem lactonization after quinone methide alkylation of a phosphodiester. The isolation of stable, fully characterized products has been imperative to the development of useful synthetic methodology from this research. This has now been accomplished.19A A variety of ester derivatives have been synthesized to study the requirements for trapping the trialkylphosphate through lactonization.
Characterizable quinone methide intermediates are prepared via Ag2O or PbO2 oxidation. It proved necessary to synthesize derivatives with an oxygen at the ortho-position (catechol derivatives, 10a, FIG. 5A) to exclude the formation of ortho-quinone methides upon oxidation if the esters were tethered through a methylene at the ortho-position (10b, R2=H, H, FIG. 5A). Attempts at making secondary or tertiary substituted tethers at the ortho-position (10b, R2=H, CH3 or CH3, CH3, FIG. 5A) resulted in facile lactonization (gem-dialkyl effect) circumventing oxidation to the quinone methide. These catechol derivatives may provide a beneficial modification to the ATAR design. An oxygen at the ortho-position of a para-quinone methide appears to increase the reactivity of the quinone methide towards nucleophilic addition.45A,46A The ortho-oxygen is expected to affect the rate of the lactonization reaction and the stability of the lactone product towards hydrolysis. This catechol-type system (10a) will be compared to the corresponding phenol system with an ortho-alkyl tether (10b). The quinone methide from the latter systems will be formed by 1,6-elimination of a benzylic leaving group.
The ester derivatives were made in five steps from 2,4-dimethylphenol.47 These esters include three classes: high, intermediate and low reactive derivatives (see table insert of FIG. S3A). The high reactive esters lactonized to afford 12 under the mildly basic conditions of oxidation. The low reactive derivatives were oxidized to the corresponding quinone methide 13 and underwent dibenzylphosphate addition; however, were not able to lactonize under the alkylation conditions. The intermediate reactive derivatives were successfully oxidized to para-quinone methide intermediate 13, alkylated the dibenzylphosphate to 14, and lactonized to afford trapped trialkylphosphate product 15 (FIG. S3A).
This now provides a useful synthetic approach towards the covalent functionalization of phosphodiesters. The key, fundamental reactions of the designed ATAR reagent have been successfully demonstrated. The details of these investigations are being submitted for publication.19A The alkylation of nucleotide derivatives are presently being studied having obtained enough of various required dinucleotides for NMR analysis of the phosphodiester alkylation.48A 
(6) Quinone methide formation through photolytic-initiated 1,6-elimination followed by phosphodiester alkylation. After significant effort, the conditions necessary for photolytic removal of a protecting group followed by 1,6-elimination to afford the para-quinone methide and reaction with dibenzylphosphate has been accomplished. Although there are numerous reports of reactions which occur through the presumed formation of quinone methide intermediates by 1,6-elimination processes,26A,49A,50A to our knowledge this is the first case of caged, photolytically-activated p-quinone methide formation via elimination with characterization.39A,51A 
Multiple derivatives have been synthesized as discussed below (point 8, FIG. T2A). The first successful reaction was accomplished using 16 (FIG. S4A). Photolysis of 16 (150 W xenon arc lamp, BiCl3/HCl filter,52A ambient) was monitored by 1H NMR in CDCl3 with either: (A) one equivalent of Ag+(BnO)2PO2xe2x88x92 salt, or (B) one equivalent of (i-Pr)2EtNH+(BnO)2PO2xe2x88x92 salt. After photolysis for one hour, nearly all of 16 was deprotected to form a mixture of phenol 17, quinone methide 7 and trialkylphosphate 8 in approximately 2:1:1 ratio, respectively. This photolytic-initiated reaction did not go to completion, but appears to form an equilibrium mixture of 17:7:8 under these conditions. Note that under aqueous conditions for which the ATAR is being designed to operate, the chloride will not be a competitive nucleophile, and will rapidly diffuse away from the quinone methide. Preliminary experiments having water present show no sign of equilibrium back to 17. This experiment did not have the benefit of an intramolecular trap to drain off the kinetic preferred trialkylphosphate or the assistance of a phosphate-directing guanidinium group.
(7) Hydrolytic stability of the quinone methide precursor and photolytic stability of the quinone methide intermediate and the trialkylphosphate product. Experiments have demonstrated the benefit of a carbonate protected phenol (e.g., 16, FIG. S4A) for greatly increasing the stability of the benzylchloride. Hydrolysis of quinone methide precursors has been a problem with many quinone methide-based, biologically reactive molecules.26A,43A,49A The carbonate protected 16 has shown no sign of hydrolysis at 25xc2x0 C. in 33% D2O/CD3CN for two days. Related benzyl protected derivatives hydrolyze relatively rapidly.
Due to the precedent for photolytic-induced homolytic reactions occurring with derivatives containing benzylic leaving groups,53A and the appearance of various byproducts in earlier reactions attempted, an investigation of the photolytic stability of the intermediate quinone methide and the trialkylphosphate product was undertaken. Pre-formed quinone methide 7 was photolyzed under conditions used to afford 8 above (FIG. S4A), and no reaction was evident by 1H NIMR analysis after 3 hours. Similarly, trialkylphosphate 8 was photolyzed under the same conditions and showed no sign of reaction after 3 hours.
(8) Studies of various combinations of photolabile protecting groups with different benzylic leaving groups for quinone methide formation. Successful conditions for producing identifiable quinone methide derivatives through photolytic-initiated 1,6-elimination reactions have now been realized.39A This led to the synthesis of derivatives containing either the o-nitrobenzyl (NB), the a-methyl-3,4-dimethoxy-2-nitrobenzylcarbonate (DMNBC) or the dimethoxybenzoin carbonate (DMBC) protecting group54A,55A with a variety of leaving groups at the benzylic position (FIG. T2A).56A These derivatives are being examined to determine effects of the different substituents on their hydrolytic stability,39A,43A the rates of quinone methide formation and the alkylation reaction rates.45,49 These particular derivatives allow correlation with other systems being used to study the quinone methide alkylation reaction and the lactonization reaction.
(9) Competitive guanidine cyclization: Phosphotriester formation with a quinone methide and a phosphodiester-ethylguanidinium salt. Apossible guanidine 5-exo-trig cyclization on the quinone methide of the designed ATAR was realized. This potential competition is under examination and approaches to prevent it, if necessary, are being developed (see 4.1). Initial results57A and a thorough literature search suggest this may be negligible58A,59A 
The initial analysis looked at the effect of ethylguanidinium on the dibenzylphosphate alkylation reaction with quinone methide 7. As shown in scheme 5, 0.5 equivalents of ethylguanidinium-dibenzylphosphate salt (18) in 8:1 DMSO-d6D2O was added to a solution of quinone methide 7 in CDCl3. Due to solubility problems, NMR integration shows approximately 0.3 equivalents of 18 remained in solution. Within 30 minutes at ambient temperature, the same equilibrium of 7:8 was apparent which was formed without the ethylguanidinium present (i.e., 2:1 of 7:8, FIG. S2A). Normalizing the reaction to the total amount of 18 present (0.3 equiv.), a 2:1 ratio of the equilibrium mixture of quinone methide 7 to trialkylphosphate 8 appears as an overall 10% formation of 8 by 1H NMR integration. Again, the presence of D2O had no effect on the kinetic formation of 8; however, with no intramolecular trap to drain off the kinetically formed 8, over the next several hours the presence of benzyl alcohol increased. At no point in the reaction was there any evidence of the ethyl guanidine adding to the quinone methide.60A,61A 
Although this experiment examined an intermolecular reaction and the competitive cyclization reaction in the ATAR will be intramolecular, it should be realized that there was a 1:1 ratio of the phosphate and guanidine present for reaction with the quinone methide in this experiment. In the ATAR, the complexation of the guanidinium with the phosphodiester will similarly result in a 1:1 ratio of the two components in proximity of the quinone methide. Effects of hydrogen bonding lowering the nucleophilicity of the phosphate while increasing the nucleophilicity of the guanidine were present equally in the above experiment as they will be in the ATAR-DNA alkylation reaction so these effects would also still result in the expected preference for phosphodiester alkylation.
4. Methods and Procedures
Completion of the model studies described above and the following will accomplish the design optimization of the individual ATAR components. Studies with increasingly more complex systems are beginning to coordinate the reactions into a functional derivative. Although all the studies can not be delineated, the synthesis of more complex derivatives represents the approach to be used to prepare derivatives for optimization of each component incorporated into the ATAR.
4.1. Investigations of quinone methides with tethered guanidine functional groups. Model systems are being investigated to determine the effect of a tethered guanidinium in directing the phosphodiester alkylation reaction. Our initial efforts have focused on the use of two commercially available amines: tyramine and octopamine, which were converted to their respective guanidinium-phosphate salt derivatives 18 and 19 (FIG. 6A).62A Oxidation of 18 (and a bis-Boc guanidine derivative) has not succeeded using Ag2O, PbO2, or DDQ. The inability to oxidize p-cresol suggests the 2,6-dimethyl derivative may be necessary. We recently accomplished a Stille coupling in a related 2,6-diiododerivative63A and are preparing 20 for quinone methide formation through oxidation. The 1,6-elimination of 19 to afford quinone methide has yet to succeed using acidic thermolysis. Derivative 21 is being prepared for more facile 1,6-elimination.64A 
The experiment reported above (FIG. S5A) suggests that cyclization of guanidine on the quinone methide may not occur with the guanidinium-phosphate salt complex. Should this occur, adjusting the nucleophilicity of the guanidine should alleviate this possible competition. Based on reported pKa values for various substituted guanidines,8A,65A a phenyl guanidine (10.8) is sufficiently less basic than a methyl guanidine (14.1). The nucleophilicity should be similarly weakened while still favoring the guanidinium form. Phenyl-substituted guanidines 20 and 21 (Rxe2x80x2xe2x80x3=Ph, FIG. 6A) are being synthesized66A to examine this effect.67A 
4.2. Incorporating a proton shuffle into the ATAR. Some preliminary experiments suggest incorporating a tertiary methylamine into the tether ortho to the phenol may help facilitate the ATAR reactivity. Having an estimated pKa of 9.8,8A this will act as a proton shuttle to assist quinone methide formation through deprotonation for 1,6-elimination, the conjugate acid will help to activate the quinone methide through reprotonation, and it will assist in the lactonization reaction by deprotonation of the phenol. This will have little competition with guanidine protonation, so is expected to show no deleterious effects. Analysis of this design feature will be investigated in model systems incorporating this modification. These will be synthesized using methods shown in the total syntheses below. If this modification proves unnecessary, the total syntheses below will be simplified, but will be shown with the amine to exemplify the more challenging approach.
4.3. Syntheses of functionalized ATAR model systems. Much of the chemistry for synthesizing ATAR derivatives is being developed through the various model system studies. Three different fully functionalized DNA-synthesizer machine-ready ATAR derivatives may be synthesized. The three systems are shown below based on the position of the guanidinium group. Note that each system has at least one tautomer where phosphate addition will be more favored. The flexible, non-static nature of these non-covalent interactions should be realized.68A (FIG. 7A)
(A) An ATAR derivative with the guanidine at the exocyclic methylene of the quinone methide. DNA synthesizer machine-ready derivative 25 will be prepared in ten steps from octopamine (22,FIG. S6A). Most of the key steps have already proven effective in model studies. The synthesis involves a Rathke guanylation,66A and ortho-bromination69A followed by iodination70A to make 23 for the Stille allylation selectively with the more reactive aryliodide.71A This will be converted to the amine through ozonolysis72A and reductive amination.73A The Heck reaction with acrylic acid will afford 24.74A The diimide reduction75A for reducing the alkene has already been accomplished in a model study without affecting the nitrobenzyl group.
(B) An ATAR derivative with a meta-benzylic guanidine substituent. A machineready derivative having the guanidine at the meta-benzylic position will be synthesized using a more convergent approach with a Diels-Alder reaction as the key step. A highly functionalized Danishefsky-type diene76A will be synthesized in four steps from the dianion of acetoacetic amide 26 (FIG. S7A)77A Dianion akylation78A will afford 27 which will be reduced to the aldehyde79A and converted to diene 28 by the standard approach.80A 
There is good precedent for the success of the cycloaddition of diene 28 with allene 29 to produce phenol 30 (FIG. S8A).81A Phenol 30 will be converted to machine-ready derivative 35 in 11 steps. Resonance effect allows for the selective hydrolysis of the benzylic methyl ester of 30.82A A tandem Curtius rearrangement-Rathke reaction with in situ trapping of the amine as the protected guanidine66 will be examined.83A The B-trimethylsilylethanesulfonyl (SES) protected guanidine84A will be stable throughout the synthesis, but cleaved with fluoride ion after solid-phase synthesis without hydrolyzing the carbonate (MDNB) group on the phenol. The final conversion of the methyl-trimethylsulfide to the carboxylic acid will be accomplished as in Schreiber""s total synthesis of cyclotheonamide B with related functionality in the molecule.85A 
(C) An ATAR derivative with a meta-guanidine substituent. If validated in model studies, a machine-ready derivative having the guanidine directly on the benzene ring will be prepared by one of two approaches. (1) A simple modification of the above approach using methyl acetylenedicarboxylate as the dienophile.80A (2) A directed ortho-lithiation starting from 4-aminosalicylic acid taking advantage of a MEM86A and Boc87A protecting group to assure regioselectivity.88A 
4.4. Nucleotide alkylation studies. Prior to attaching the phosphodiester alkylating reagents to oligonucleotides for site-selective delivery, reactions will be run using the appropriate derivative synthesized above89A with dinucleotides for complete product characterization and reaction optimization.48A,90A Dodecanucleotides will then be examined for peralkylation. The presence of the guanidinium groups in the alkylated polymer should maintain the water solubility of the trialkylphosphate product.91A The reversal of charge will reverse the polarity necessary for PAGE analysis or slow the migration of partially alkylated oligonucleotides.36A Assessing the degree of alkylation of the whole oligonucleotides can be determined qualitatively by gel migration analyses using PAGE on the 5xe2x80x2-32p labeled oligos.92A,93A,94A Initial digestion of the oligonucleotides from the alkylation reaction with snake venom phosphodiesterase and/or calf intestine alkaline phosphatase will result in cleavage of the oligonucleotides only at unmodified phosphodiester linkages, as phosphotriester linkages are known to be stable to degradation.16A HPLC analysis of the resulting products for the degree of alkylation will assess if there was any regioselectivity.95A The degree of alkylation will be further analyzed by high resolution mass spectrometry (MALDITOF). NMR analysis will be attempted to determine chemo- and possibly diastereo-selectivity""s and for assessing the structural characteristics of the products.96A Crystallization of the products for x-ray diffraction analysis may succeed with the guanidinium group incorporated.
4.5. Oligonucleotide attachment. The delivery oligonucleotide of desired sequence will be synthesized on an automated DNA synthesizer according to standard protocol.97A Modifying oligonucleotides with any desired linker is common practice.17A A C12 chain length phosphoramidite will be synthesized98A and attached to the oligonucleotide.96A,99A A standard esterification reaction will attach the ATAR derivative as synthesized above to the linker-OH on the solid support.100A Mild alkaline hydrolysis of the TFA-protected amine101A will allow the attachment of the desired reporter group, drug agent, or protein conjugate. Some examples of reporter groups for initial studies of oligonucleotide modification using this ATAR include: fluorescein-5-isotholocyanate,102A the EDTA.Fe(II) moiety,103A and tris(2,2xe2x80x2-bipyridine)ruthenium (II) (Ru(bpy)32.104A Complete protecting group removal and cleavage from the solid-support105A will afford a functional ATAR.106A Drugs6A,10A and derivatized proteins7A,11A will be attached similarly. The resulting ATAR derivatives will be purified by HPLC. The derivatized oligonucleotides will be characterized by enzymatic digestion and HJPLC analysis against coinjections of standard solutions of the nucleoside components and a reagent standard with the attached linker. An exact mass will also be obtained. ATAR attachment will be confirmed by UV analysis.107A 
Affinity cleavage experiments canbe conducted with the EDTA.Fe(II) group attached to the ATAR for analysis of labeling both single- and double-strand target DNA according to established methods.3A,108A Analysis of the diffusible cleavage pattern on the DNA to which the ATAR has been delivered will allow assessment of the structural characteristics of the ATAR-DNA interactions.
4.6. Future studies. The ability to modify nucleosomal DNA will allow various crosslinking and autocleavage investigations to be conducted for enhancing our understanding of DNA-protein interactions in the chromatin.4A Transcriptional regulation will be studied by using the ATAR to attach transcriptional regulator GCN5p7A and other transcriptional activators to selected sites on DNA.7A It will also be of interest to study how drugs known to bind to, and react with DNA will be affected by their covalent attachment through the ATAR.109A 
(1A) For a complete resource covering many aspects of the human genome see the Genome Database (GDB) hosted at Johns Hopkins University (http://gdbwww.gdb.orgf): Fasman, K. H.; Letovsky, S. I.; Li, P.; Cottingham, R. W.; Kingsbury, D. T. xe2x80x9cThe GDB Human Genome Database Anno 1997,xe2x80x9d Nucleic Acids Res. 1997, 25, 72-80.
(2A) (a) Leonetti, J. P.; Degols, G.; Clarenc, J. P.; Mechti, N.; Lebleu, B. xe2x80x9cCell Delivery and Mechanism of Action of Antisense Oligonucleotides,xe2x80x9d Prog. Nucleic Acids Res. McI. Biol. 1993, 44, 143-66. (b) Zon, G. xe2x80x9cBrief Overview of Control of Genetic Expression by Antisense Oligonucleotides and In Vivo Applications,xe2x80x9d Molec. Neurobiol. 1995, 10, 219-29.
(3A) (a) Thuong, N. G.; Hxc3xa9lxc3xa9ne, C. xe2x80x9cSequence-Specific Recognition and Modification of Double-Helical DNA Oligonucleotides,xe2x80x9d Angew. Chem. Int. Ed. Engl. 1993, 32,666-90. (b) Dervan, P. R. xe2x80x9cReagents for the Site-Specific Cleavage of Megabase DNA,xe2x80x9d Nature 1992, 359, 87. (c) Dervan, P. R. xe2x80x9cDesign of Sequence Specific DNA Binding Molecules,xe2x80x9d Science 1986, 232, 464.
(4A) A system such as being proposed will be particularly valuable for providing information at the molecular level in multi-protein complexes interacting with nucleic acids. These would include the complex protein-nucleic acid interactions of the chromatin involved in chromosome condensation-decondensation, DNA replication, transcription, transcription regulation and DNA repair. Molecular level details in such complex systems are difficult to achieve by existing biochemical techniques and advances in molecular biology require innovative approaches to begin to develop a more thorough molecular level understanding of the chromosomal protein machinery. For example, the presumed role of histone H1 in transcriptional repression might be studied by site-specifically modifying a target DNA binding sequence with crosslinking and redox activated cleaving functionality for mapping DNA-histone H1 interactions: (a) Paranjape,S. M.; Kamakaka, R. T.; Kadonaga, J. T. xe2x80x9cRole of Chromatin Structure in the Regulation of Transcription by RNA Polymerase II,xe2x80x9d Annu. Rev. Biochem. 1994, 63,265-97. (b) Felsenfeld, G. xe2x80x9cChromatinas an Essential Part of the Transcriptlonal Mechanism,xe2x80x9d Nature 1992, 355, 219-24. (c) Halmer, L.; Gruss, C. xe2x80x9cInfluence of Histone H1 on the in vitro Replication of DNA and Chromatin,xe2x80x9d Nucleic Acids Res. 1995, 23, 773-78.
(5A) The ability to label a hybridization-recognized sequence of DNA should afford an efficient approach to genetic diagnostics from blood samples. The chemistry being developed will allow the efficient synthesis of multiply-labeled oligonucleotides which can he used for genetic diagnostics by methods such as fluorescence in-situ hybridization (FISH). (a) Brenner, M.; Dunlay, T. xe2x80x9cFluorescence In vitro Hybridization. Hardware and Software Implications in the Research Laboratory,xe2x80x9d Amer. Laboratory 1995, 55-58. (b) For lanthanide-labeled DNA probes, see: Lxc3x6vgren, T.; Hurskainen, P.; Dahlxc3xa9n, P. in Nonisotopic DNA Probe Techniques, Kricka, L. J., Ed.; Academic Press, Inc.: San Diego; 1992, pp. 227-274.
(6A) A particularly appealing application would be in the area of site-specific drug delivery to genetic targets. The non-specific deliterious effects of chemotherapy on healthy cells could be alleviated using such a system to covalently deliver an antitumor antibiotic directly to a target DNA sequence.
(7A) Innovative experiments which could be attempted with such a system include modifying nucleosomal DNA with transcriptional regulator GCN5p. This transcriptional regulator functions as a complex with two other proteins (ADA2p and ADA3p). It has recently been found to be a histone acetyltransferase. Histone hyperacetylation is thought to facilitate transcription by chromatin disruption, but it is not clear whether the histone hyperacetylation is a result of chromatin disruption during the transcription process, or an initiator. This regulatory complex with GCN5p is recruited to a specific gene through interactions with other DNA binding transcripttion factors. A system such as being proposed would allow site-specific delivery of this regulatory protein to a particular chromatin site. This could then recruit the regulatory complex and other transcription factors and thereby initiate transcription of a selective gene. Obviously, such an approach could be used to regulate many cellular functions through selective control of genetic transcription. For leading references see: (a) Wolfe, A. P.; Pruss, D. xe2x80x9cTargeting Chromatin Disruption: Transcription Regulators that Acetylate Histones,xe2x80x9d Cell 1996, 84, 817-19. (b) Ptashne, M.; Gann, A. xe2x80x9cTranscriptional activation by recruitment,xe2x80x9d Nature 1997, 386, 569-77.
(8A) Although only a very crude measure, estimated pKa values calculated from the effects of various related substituted derivatives suggest that the proton shuttle processes proposed should occur as drawn in scheme 1. The relevant pKa values (H2O, 25xc2x0 C.) include: 2,4,6-trimethylphenol (10.88), 3-aminophenol (9.83), m-cresol (10.00), phenol (9.99), Et2MeN (10.4), phenethylamine (9.83), ethylamine (10.63), guanidine (14.38), methylguanidine (14.1), phenylguanidine (10.77). From these values, pKa estimates in the ATAR may be approximated assuming additivity of substituent effects. The approximated pKa would be: 10.7 for the phenoxide with a 3-amino group on the ring (the effect of a 3-amino on the pKa of phenol is xcex94pKa=xe2x88x920.16; thus approximating from the pKa of 2,4,6-trimethylphenol=10.88-0.16=10.7, other values are determined in a similar way), 10.9 for the phenoxide with the 3-benzylic-amino group, 9.8 for the tertiary amine, 10.8 for the guanidine directly substituted on the arene ring, 10.5 for the benzylic guanidine with a phenyl substituent. The pKa values are from Lange""s Handbook of Chemistry, Dean, J. A, Ed.; McGraw-Hill: N.Y. 1992, 14th edition.
(9A) Reporter groups would include fluorescent probes (For example, see: Haugland, R. P. Molecular Probes: Handbook of Fluorescent Probes and Research Chemicals, Larison, K. D., Ed.; 1995-1997, 6th Edition, Molecular Probes, Inc., Eugene, Oreg.), probes used for recognition of a specific species such as biotin/avidin and antibodies, luminescent probes, probes which are chemically or redox reactive, radionuclear probes, and magnetic moieties ((a) Wilbur, D. S. xe2x80x9cRadiohalogenation of Proteins: An Overview of Radionuclides, Labeling Methods, and Reagents for Conjugate Labeling,xe2x80x9d Bioconjugate Chem. 1992, 3, 433-471. (b) Peters, K.; Richards, F. M. xe2x80x9cChemical Crosslinking: Reagents and Problems in Studies of Membrane Structure,xe2x80x9d Ann. Rev. Biochem. 1977, 46, 523-51. (c) Ji, T. H. xe2x80x9cThe Application of Chemical Crosslinking for Studies on Cell Membranes and the Identification of Surface Reporters,xe2x80x9d Biochim. Biophys. Acta 1979, 559, 39-69.).
(10A) For examples of drugs which could be readily attached, see a listing of anticancer agents along with associated references in: Calbiochem Biochemical and Immunochemical 1996/97 Catalog, p. 539, San Diego, Calif.
(11A) For an example of protein conjugation to an oligonucleotide for directing nuclease activity, see: Pei, D.; Corey, D. R.; Schultz, P. G. xe2x80x9cSite-specific Cleavage of Duplex DNA by a Semi-synthetic Nuclease via Triple-helix Formation,xe2x80x9d Proc. Nat. Acad. Sci. USA 1990, 87, 9858.
(12A) For conventional synthesis of phosphate modified oligonucleotides, see: (a) Hayakawa, Y.; Hirose, M.; Hayakawa, M.; Noyori, R. xe2x80x9cGeneral Synthesis and Binding Affinity of Position-Selective Phosphonodiester- and Phosphotriester-Incorporated Oligodeoxyribonucleotides,xe2x80x9d J. Org. Chem. 1995, 69, 925-30. (b) Hayakawa, Y.; Wakabayashi, S.; Kato, H.; Noyori, R. xe2x80x9cThe Allylic Protection Method in Solid-Phase Oligonucleotide Synthesis. An Efficient Preparation of Solid-Anchored DNA Oligomers,xe2x80x9d J. Am. Chem. Soc. 1990, 112, 1691-96. (c) Kujipers, W. H. A.; Huskens, J.; Koole, L. H.; van Boeckel, C. A. A. xe2x80x9cSynthesis of Well-Defined Phosphate-Methylated DNA Fragments: the Application of Potassium Carbonate in Methanol as Deprotecting Reagent,xe2x80x9d Nucleic Acids Res. 1990, 18, 5197-205. (d) Alul, R. H.; Singman, C. N.; Zhang, G.; Letsinger, R. L. xe2x80x9cOxalyl-CPG: A Labile Support for the Synthesis of Sensitive Oligonucleotide Derivatives,xe2x80x9d Nucleic Acids Res. 1991, 19, 1527-32. (e) Froehler, B. C. xe2x80x9cDeoxynucleoside H-Phosphonate Diester Intermnediates in the Synthesis of Internucleotide Phosphate Analogues,xe2x80x9d Tetrahedron Left. 1986, 27, 557-78.
(13A) For additional synthesis reviews see: (a) Beaucage, S. L.; Iyer, R. P. xe2x80x9cAdvances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach,xe2x80x9d Tetrahedron 1992, 48, 2223-2311. (1,) Hobbs, J. B. xe2x80x9cNucleotides and Nucleic Acids,xe2x80x9d Organophosphorus Chemistry 1990, 21, 201-321. (c) Sonveaux, E. xe2x80x9cThe Organic Chemistry Underlying DNA Synthesis,xe2x80x9d Bioorg. Chem. 1986, 14, 274-325.
(14A) Studies using phosphate triester modified oligos for duplex structure studies with RNA and DNA: (a) Letsinger, R. L.; Bach, S. A.; Eadie, J. S. xe2x80x9cEffects of Pendant Groups at Phosphorus on Binding Properties of d-ApA Analogues,xe2x80x9d Nucleic Acids Res. 1986, 14,3487-99. (b) Summers, M. F.; Powell, C.; Egan, W.; Byrd, R. A.; Wilson, W. D.; Zon, G. xe2x80x9cAlkyl Phosphotriester Modified Oligodeoxyribonucleotides. VI. NMR and UV Spectroscopic Studies of Ethyl Phosphotriester (Et) Modified Rp-Rp and Sp-Sp Duplexes, {d[GGAA(Et)TTCC]}2xe2x80x9d Nucleic Acids Res. 1986, 14, 7421-37. (c) Pramanik, P.; Kan, L. xe2x80x9cNMR Study of the Effect of Sugar-phosphate Backbone Ethylation on the Stability and Conformation of DNA Double Helix,xe2x80x9d Biochemistry 1987, 26, 3807-12. (d) Koole, L. H.; van Genderen, M H Buck, H. M. xe2x80x9cA Parallel Right-Handed Duplex of the Hexamer d(TpTpTpTpTpT) with Phosphate Triester Linkages,xe2x80x9d J. Am. Chem. Soc. 1987, 109, 3916-21 *[The synthetic chemistry and hybridization data reported in this 1987 paper differed from that described later and subsequently retracted by Buck, H. M.; Moody, H. M.; Quaedflieg, P. J. L. M.; Koole, L. H.; van Genderen, M. H. P.; Smit, L. Jurriaans, S. Geelen, J. L. M. C.; Goudsmit, J. xe2x80x9cInhibition of HIV-1 Infectivity by Phosphate-Methylated DNA: Retraction,xe2x80x9d Science 1990, 250, 125-26 (also see: Maddox, J. xe2x80x9cDutch Cure for AIDS is Discredited,xe2x80x9d Nature 1990, 347, 411).]. (e) Quaedflieg, P. J. L. M.; Koole, L. H.; van Genderen, M. H. P.; Buck, H. M. xe2x80x9cA structural Study of Phosphate-methylated d(CpG)n and d(GpC)n DNA oligomers. Implications of Phosphate Shielding for the Isomerization of B-DNA into Z-DNA,xe2x80x9d Recl. Trav. Chim. Pay-Bas 1989, 108, 421-23. (f) Quaedflieg, P. J. L. M.; Broeders, N. L. H. L.; Koole, L. H.; van Genderen, M. H. P.; Buck, H. M. xe2x80x9cConformation of the Phosphate-methylated DNA Dinucleotides d(Cp,C) and d(TpC). Formation of a Parallel Miniduplex Exclusively for the S-Configuration at Phosphorus,xe2x80x9d J. Org. Chem. 1990, 55, 122-27. (g) Quaedflieg, P. J. L.; van der Heiden, A. P.; Koole, L. H.; Coenen, A. J. J. M.; van der Wal, S.; Meijer, E. M. xe2x80x9cSynthesis and Conformational Analysis of Phosphate-methylated RNA Dinucleotides,xe2x80x9d J. Org. Chem. 1991, 56, 5846-59.
(15A) Using modified triester phosphate oligos as probes for elucidating specific interactions with proteins: (a) Weinfeld, M.; Drake, A. F.; Saunders, J. K.; Paterson, M. C. xe2x80x9cStereospecific Removal of Methyl Phosphotriesters from DNA by an Escherichia coli ada+Extract,xe2x80x9d Nucleic Acids Res. 1985, 13, 7067-77. (b) Gallo, K. A.; Shao, K; Phillips, L. R.; Regan, J. B.; Kozielkiewicz, M.; Uznanski, B.; Stec, W. J.; Zon, G. xe2x80x9cAlkyl Phosphotriester Modified Oligodeoxyribonucleotides. V. Synthesis and Absolute Configuration of Rp and Sp, Diastereomers of an Ethyl Phosphotriester (Et) Modified EcoRI Recognition Sequence, d[GGAA(Et)TTCC]. A Synthetic Approach to Regio and Stereospecific Ethylation-interference Studies,xe2x80x9d Nucleic Acids Res. 1986, 14, 7405-20. (c) Kbziolkiewicz, M.; Stec, W. J. xe2x80x9cApplication of Phosphate-backbone-modified Oligonucleotides in the Studies on EcoRI Endonuclease Mechanism of Action,xe2x80x9d Biochemistry 1992, 31, 9460-66.
(16A) (a) Miller, P. S.; Fang, K. N.; Kondo, N. S.; Ts""O, P.O P. xe2x80x9cSynthesis and Properties of Adenine and Thymidine Nucleoside Alkyl Phosphotriesters, the Neutral Analogs of Dinucleoside Monophosphates,xe2x80x9d J. Am. Chem. Soc. 1971, 93, 6657-65. (b) Milrer, P. S.; Barrett, J. C.; Ts""O, P.O.P. xe2x80x9cSynthesis of Oligodeoxyribo-nucleotide Ethyl Phosphotriesters and Their Specific Complex Formation with Transfer Ribonucleic Acid,xe2x80x9d Biochemistry 1974, 13, 4887-96 (and the following paper in that journal as well). (c) Pless, R. C.; Ts""O, P.O.P. xe2x80x9cDuplex Formation of a Nonionic Oligo(deoxythymidylate) Analogue [Heptadeoxythymidylyl-(3xe2x80x2-5xe2x80x2)-deoxythymidine Heptaethyl Ester (d-[Tp(Et)]7T)] with Poly(deoxyadenylate. Evaluation of the Electrostatic Interaction,xe2x80x9d Biochemistry 1977, 16, 1239-50. (d) Miller, P. S.; Braiterman, L. T.; Ts""O, P.O.P. xe2x80x9cEffects of a Trinucleotide Ethyl Phosphotriester, Gmp(Et)Gm(Et)U, on Mammalian Cells in Culture,xe2x80x9d Biochemistty 1977, 16, 1988-96. (e) Petrenko, V. A.; Pozdnyakov, P. I.; Kipriyanov,S. M.; Boldyrev, A. N.; Semyonova, L. N.; Sivolobova, G. F. xe2x80x9cSite-localized Mutagenesis Directed by Phosphotriester Analogs of Oligonucleotides,xe2x80x9d Bioorg. Khim. 1986, 12, 1088-1100. (f) Asseline, U.; Barbier, C.; Thuong, N. T. xe2x80x9cOligothymidylates Comportant La Structure Alternee AIkylphosphotriester-phosphodiester et Lies de Facon Covalente a un Agent Intercalant,xe2x80x9d Phosphorus Sulfur 1986, 26, 63-73. (g) Marcus-Sekura, C. J.; Woerner, A. M.; Shinozuka, K.; Zon, G.; Quinnan, Jr., G. V. xe2x80x9cComparative Inhibition of Cloramphenicol Acyltransferase Gene Expression by Antisense Oligonucleotide Analogs Having Alkyl Phosphotriester, Methylphosphonate and Phosphorothioate Linkages,xe2x80x9d Nucleic Acids Res. 1987, 15, 5749-63. (h) see ref. 6a. (i) Koole, L. H.; van Genderen, MIH.P.; Reiniers, R. G.; Buck, H. M. xe2x80x9cEnhanced Stability of a Watson and Crick DNA Duplex Structure by Methylation of the Phosphate Groups in One Strand,xe2x80x9d Proc. K. Ned. Akad. Wet. B 1987, 90, 41-6.* (j) Petrenko, V. A.; Kipriyanov, S. M.; Boldyrev, A. N.; Pozdnyakov, P. I. xe2x80x9cMutagenesis Directed by Phosphotriester Analogues of Oligonucleotides: a Way to Site-specific Mutagenesis In Vivo,xe2x80x9d FEBS Lett. 1988, 23,109-12. (k) Durand, Maunizot, J. C.; Asseline, U.; Barbier, C.; Thuong, N. T.; Hxc3xa9lxc3xa9ne, C. xe2x80x9cOligothymidylates Covalently Linked to an Acridine Derivative and with Modified Phosphodiester Backbone: Circular Dichroism Studies of Their Interactions with Complementary Sequences,xe2x80x9d Nucleic Acids Res. 1989, 17, 1823-36.
(17A) For recent reviews see: (a) Zon, G. xe2x80x9cBrief Overview of Control of Genetic Expression by Antisense Oligonucleotides and In Vivo Applications,xe2x80x9d Mol. Neurobiology 1995, 10, 219-29. (b) Kiely, T. S. xe2x80x9cRecent Advances in Antisense Technology,xe2x80x9d Ann. Rep. Med. Chem. 1994, 29,297-306. (c) Stein, C. A.; Cheng, Y.-C. xe2x80x9cAntisense Oligonucleotides as Therapeutic Agents-Is the Bullet Really Magic,xe2x80x9d Science 1993, 261, 1004-11. (d) Varma, R. S. xe2x80x9cSynthesis of Oligonucleotide Analogues with Modified Backbones,xe2x80x9d SYNLETT 1993, 621-37. (e) Beaucage, S. L.; Iyer, R. P. xe2x80x9cThe Functionalization of Oligonucleotides Via Phosphoramidite Derivatives,xe2x80x9d Tetrahedron 1993, 49, 1925-63. (f) Toulmxc3xa9, J. J. in Antisense RNA and DNA; Murray, J. A. H., Ed.; Wiley, Inc.: New York, 1992, pp 175-94. (g) Englisch, U.; Gauss, D. H. xe2x80x9cChemically Modified Oligonucleotides as Probes and Inhibitors,xe2x80x9d Angew. Chem. Int. Ed. EngI. 1991, 30, 613-722. (h) Uhlmann, E.; Peyman, A. xe2x80x9cAntisense Oligonucleotides: A New Therapeutic Principle,xe2x80x9d Chem. Rev. 1990, 90, 543-84. (i) Goodchild, J. xe2x80x9cConjugates of Oligonucleotides and Modified Oligonucleotides: A Review of Their Synthesis and Properties,xe2x80x9d Bioconjugate Chem. 1990, 1, 165-87. (j) Hxc3xa9lxc3xa9ne, C.; Toulmxc3xa9, J.-J. xe2x80x9cSpecific Regulation of Gene Expression by Antisense, Sense, and Antigene Nucleic Acids,xe2x80x9d Biochim. Biophys. Acta 1990, 1049, 99-125. (k) Goodchild, J. xe2x80x9cInhibition of Gene Expression by Oligonucleotides,xe2x80x9d in Oligonucleotides: Antisense Inhibitors of Gene Expression; Cohen, 1.5., Ed.; McMillan Press: London, 1989, pp. 53-771 (l) Zon, G. xe2x80x9cOligonucleotide Analogues as Potential Chemotherapeutic Agents,xe2x80x9d Pharm. Res. 1988, 5, 539-49. (m) Stein, C. A.; Cohen, J. S. xe2x80x9cOligonucleotides as Inhibitors of Gene Expression: a Review,xe2x80x9d Cancer Res. 1988, 48, 2659-68. (n) Miller, P. S.; Ts""O, P.O.P. xe2x80x9cOligonucleotide Inhibitors of Gene Expression in Living Cells: New Opportunities in Drug Design,xe2x80x9d Annu. Rep. Med. Chem. 1988, 23, 295-304. (o) Miller, P. S.; Agris, C. H.; Blake, K. R.; Murakami, A.; Spitz, S. A.; Reddy, M. P.; Ts""O, P.O.P. xe2x80x9cNonionic Oligonucleotide Analogs as New Tools for Studies on the Structure and Function of Nucleic Acids in Living Cells,xe2x80x9d in Nucleic Acids: The Vectors of Life; Pullman, B.; Jortner, J., Eds.; D. Reidel Publishing Co.: Dordrecht, Netherlands; 1983, pp. 521-35.
(18A) A review has proposed the use of the acronym SNAIGE (Synthetic or Small Nucleic Acid Interfering with Gene Expression) as a term for describing the various approaches to genetic regulation with oligonucleotides; Leonetti, J. P.; Degols, G.; Clarenc, J. P.; Mechti, N.; Lebleu, B. xe2x80x9cCell Delivery and Mechanism of Action of Antisense Oligonucleotides,xe2x80x9d Prog. Nucl. Acid Res. 1993, 44, 143-66.
(19A) Zhou, Q.; Turnbull, K. D. xe2x80x9cPhosphotriesters from Tandem Phosphodiester Alkylation with Quinone Methides Followed by Lactonization,xe2x80x9d manuscript near completion for submission to: J. Am. Chem. Soc. 1997, 119.
(20A) Reliable Biopharmaceuticals (St. Louis, Mo.), a supplier of oligonucleotide derivatives for antisense and antigene applications, has expressed interest in this work. We are conducting preliminary experiments to determine the potential for collaborative development of synthetic methodology for oligonucleotide modification.
21A) Sigman, D. S.; Mazumder, A.; Pemn, D. M. xe2x80x9cChemical Nucleases,xe2x80x9d Chem. Rev. 1993, 93, 2295-316.
(22A) More recent examples include: (a) Jubian, V.; Dixon, R. P.; Hamilton, A. D. xe2x80x9cMolecular Recognition and Catalysis. Acceleration of Phosphodiester Cleavage by a Simple Hydrogen-Bonding Receptor,xe2x80x9d J. Am. Chem. Soc. 1992, 114, 1120-21. (b) Browne, K. A.; Bruice, T. C. xe2x80x9cChemistry of Phosphodiesters, DNA and Models. 2. The Hydrolysis of Bis(8-hydroxyquinoline) Phosphate in the Absence and Presence of Metal Ions,xe2x80x9d J. Amer. Chem. Soc. 1992, 114, 4951-58. (c) Smith, J.; Ariga, K.; Anslyn, E. V. xe2x80x9cEnhanced Imidazole-Catalyzed RNA Cleavage Induced by aBis-Alkylguanidinium Receptor,xe2x80x9d J. Am. Chem. Soc. 1993, 115,362-64. (d) Takasaki, B. K.; Chin, J. xe2x80x9cSynergistic Effect Between La(III) and Hydrogen Peroxide in Phosphate Diester Cleavage,xe2x80x9d J. Am. Chem. Soc. 1993, 115, 99337-38. (e) Hall, J. Husken, D.; Pieles, U.; Moser, H. E.; Haner, R. Chemistry and Biology 1994, 1, 185-90. (f) Bashkin, J. K.; Frolova, E. I.; Sampath, U. xe2x80x9cSequence-Specific Cleavage of HIV mRNA by a Ribozyme Mimic,xe2x80x9d J. Am. Chem. Soc. 1994, 116, 5981-82. (g)Magda, D.; Miller, R. A.; Sessler, J. L.; Iverson, B. L. xe2x80x9cSite-Specific Hydrolysis of RNA by Europium(III) Texaphyrin Conjugated to a Synthetic Oligodeoxyribonucleotide,xe2x80x9d J. Am. Chem. Soc. 1994, 116, 7439-40. (h) Linkletter, B.; Chin, I. xe2x80x9cRapid Hydrolysis of RNA with a CuII Complex,xe2x80x9d Angew. Chem. Int. Ed. Engl. 1995, 34, 472-74.
(23A) (a) Wilbur, D. S. xe2x80x9cRadiohalogenation of Proteins: An Overview of Radionuclides, Labeling Methods, and Reagents for Conjugate Labeling,xe2x80x9d Bioconjugate Chem. 1992, 3, 433-471. (b) Peters, K., Richardg, F. M. xe2x80x9cChemical Crogg-linking: Reagents and Problems in Studies of Membrane Structure,xe2x80x9d Ann. Rev. Biochem. 1977, 46, 523-51. (c) Ji, T. H. xe2x80x9cThe Application of Chemical Crosslinking for Studies on Cell Membranes and the Identification of Surface Reporters,xe2x80x9d Biochim. Biophys. Acta 1979, 559, 39-69.
(24 A) Zhang, Y.; Chen, F.-X.; Mehta, P.; Gold, B. xe2x80x9cGroove-and Sequence-Selective Alkylation of DNA by Sulfonate Esters Tethered to Lexitropsins,xe2x80x9d Biochemistry 1993, 32, 7954-65.
(25A) For reviews on quinone methides, see: (a) Volod""kin, A. A.; Ershov, V. V. Russian Chem. Rev. 1988, 57,336. (b) Gruenanger, P. in Houben-Weyl Methoden der Organischen Chemie (Vol. VII/3b) Mueller, E.; Bayer, O., Eds.; G. Thieme Verlag: Stuttgart, 1979, pp. 195-521. (c) Wagner, H.-U.; Gompper, R. in The Chemistry of Quinonoid Compounds (Vol. 1) Patai, S., Ed.; John Wiley and Sons: New York, 1974, pp. 1145-1178. (d) Turner, A. B. Quart. Rev. 1965, 18, 347.
(26A) More recent, elegant examples for biomolecule alkylation include: (a) Chatterjee, M.; Rokita, S. E. xe2x80x9cThe Role of a Quinone Methide in the Sequence Specific Alkylation of DNA,xe2x80x9d J. Am. Chem. Soc. 1994, 116, 1690-97. (b) Li, T.; Zeng, Q.; Rokita, S. E. xe2x80x9cTarget-Promoted Alkylation of DNA,xe2x80x9d Bioconjugate Chem. 1994, 5, 497-500. (c) Meyers, J. K.; Cohen, J. D.; Widlanski, T. S. xe2x80x9cSubstituent Effects on the Mechanism-Based Inactivation of Prostatic Acid Phosphatase,xe2x80x9d J. Am. Chem. Soc. 1995, 117, 11049-54. (d) Myers, J. K.; Widlanski, T. S. xe2x80x9cMechanism-Based Inactivation of Prostatic Acid Phosphatase,xe2x80x9d Science 1993, 262, 1451-53. (e) Wang, Q.; Dechert, U.; Jirik, F.; Withers, S. G. xe2x80x9cSuicide Inactivation of Human Prostatic Acid Phosphatase and a Phosphotyrosine Phosphatase,xe2x80x9d Biochem. Biophys. Res. Commun. 1994, 200, 577-83.
(27A) For reviews see: (a) Sega, G. A. xe2x80x9cA Review of the Genetic Effects of Ethyl Metianesulfonate,xe2x80x9d Mutation Res. 1984, 134, 113-42. (b) Hoffmann, G. R. xe2x80x9cGenetic Effects of Dimethyl Sulfate, Diethyl Sulfate, and Related Compounds,xe2x80x9d Mutation Res. 1980, 75, 63-129. (c) Digenis, G. A.; Issidorides, C. H. xe2x80x9cSome Biochemical Aspects of N-Nitroso Compounds,xe2x80x9d Bioorganic Chem. 1979, 8, 91-137.
(28A) (a) Swenson, D. H.; Lawley, P. D. xe2x80x9cAlkylation of Deoxyribonucleic Acid by Carcinogens Dimethylsulfate, Ethyl Methanesulfonate, N-Ethyl-N-nirosourea and N-Methyl-N-nitrosourea,xe2x80x9d Biochem. J. 1978, 171, 575-87. (a) Jensen, D. E.; Reed, D. J. xe2x80x9cReaction of DNA with Alkylating Agents. Quantitation of Alkylation by Ethylnitrosourea of Oxygen and Nitrogen Sites on Poly[dA-dT] Including Phosphotriester Formation,xe2x80x9d Biochemistry 1978, 17, 5098-107. (c) Sun, L.; Singer, B. xe2x80x9cThe Specificity of Different Classes of Ethylating Agents Towards Various Sites of HeLa DNA in vitro and in vivo,xe2x80x9d Biochemistry 1975, 14, 1795-1802.
(29A) Angle, S. R.; Arnaiz, D. O.; Boyce, l. P.; Frutos, R. P.; Louie, M. S.; Mattson-Arnaiz, H. L.; Rainier, J. D.; Turnbull, K. D.; Yang, W. xe2x80x9cFormation of Carbon-Carbon Bonds via Quinone Methide-Initiated Cyclization Reactions,xe2x80x9d J. Org. Chem. 1994, 59, 6322-6337.
(30A) Organic Synthesis, Smith, M. B.; McGraw-Hill, Inc.: New York; 1994, pp. 108-119.
(31A) Schulz, W. G.; Nieman, R. A.; Skibo, E. B. xe2x80x9cEvidence for DNA Phosphate Backbone Alkylation and Cleavage by Pyrrolo[1,2-a]benzimidazoles: Small Molecules Capable of Causing Base-Pair-Specific Phosphodiester Bond Hydrolysis,xe2x80x9d Proc. NatI. Acad. Sci. USA 1995, 92, 11854-58.
(32A) (a) Tomasz, M.; Lipman, R. xe2x80x9cAlkylation Reactions of Mitomycin C at Acid pH,xe2x80x9d J. Am. Chem. Soc. 1979, 101, 6063-67. (b) Iyengar, B. S.; Dorr, T. R.; Remers, W. A.; Kowal, C. D. xe2x80x9cNucleotide Derivatives of 2,7-Diaminomitosene,xe2x80x9d J. Med. Chem. 1986, 31, 1579-85.
(33A) Gohil, R. N.; Roth, A. C.; Day, R. A. xe2x80x9cReversible Covalent Modification of DNA,xe2x80x9d Arch. Biochem. Biophys. 1974, 165, 297-312.
(34A) Bhat, G.; Roth, A. C.; Day, R. A. xe2x80x9cExtrinsic Cotton Effect and Helix-Coil Transition in a DNA-Polycation Complex,xe2x80x9d Biopolymers 1977, 16, 1713-24.
(35A) For a thorough review, see: Hannon, C. L.; Anslyn, E. V. xe2x80x9cThe Guanidinium Group: Its Biological Role and Synthetic Analogs,xe2x80x9d Bioorg. Chem. Frontiers 1993, 3, 193-255.
(36A) For additional examples, see: (a) Blaskxc3x3, A.; Dempcy, R. O.; Minyat, E. E.; Bruice, T. C. xe2x80x9cAssociation of Short-Strand DNA Oligomers with Guanidinium-Linked Nucleosides. A Kinetic and Thermodynamic Study,xe2x80x9d J. Am. Chem. Soc. 1996, 118, 7892-99. (b) Dempcy, R. O.; Browne, K. A.; Bruice, T. C. xe2x80x9cSynthesis of the Polycation Thymidyl DNG, Its Fidelity in Binding Polyanionic DNA/RNA, and the Stability and Nature of the Hybrid Complexes,xe2x80x9d J. Am. Chem. Soc. 1993, 117, 6140. (c) Hashimoto, H.; Nelson, M. G.; Switzer, C. xe2x80x9cFormation of Chirneric Duplexes Between Zwitterionic and Natural DNA,xe2x80x9d J. Org. Chem. 1993, 58, 4194-95. (d) Hashimoto, H.; Nelson, M. G.; Switzer, C. xe2x80x9cZwitterionic DNA,xe2x80x9d .J Am. Chem. Soc. 1993, 115, 7128-34. (e) Letsinger, R. L.; Sinan, C. N.; Histand, G.; Salunkhe, M. xe2x80x9cCationic Oligonucleotides,xe2x80x9d J. Am. Chem. Soc. 1988, 155, 7128. (f) Furberg, S.; Solbakk, J. xe2x80x9cOn the Stereochemistry of the Interaction Between Nucleic Acids and Basic Protein Side Chains,xe2x80x9d Acta Chem. Scand. B 1974, 28,481-83.
(37A) Saenger, W. Principles of Nucleic Acid Structure, Springer-Verlag: New York, 1984, pp. 385-431.
(38A) Pullman, B. xe2x80x9cMolecular Mechanisms of Specificity in DNA-Antitumor Drug Interactions,xe2x80x9d in Advances in Drug Research, Testa, B., Ed.; Academic Press: London; Vol. 18, 1989, pp. 1-113.
(39A) (a) Zhou, Q.; Tumbull, K. D. xe2x80x9cEquilibrium Control in Phosphodiester Alkylation with Quinone Methides,xe2x80x9d manuscript in preparation for submission to: J. Org. Chem. 1997, 62. (b) Dyer, R. G.; Tumbull, K. D. xe2x80x9cPhotolytic-Initiated Formation of Quinone Methides for Phosphodiester Alkylation,xe2x80x9d manuscript in preparation.
(40A) Patent File number ARK0O7/97386 for xe2x80x9cB iomolecular Labelingxe2x80x9d (Apr. 11, 1997). 
(41A) Dyall, L. K.; Winstein, S. xe2x80x9cNuclear Magnetic Resonance Spectra and Characterization of Some Quinone Methides,xe2x80x9d J. Am. Chem. Soc. 1972, 94, 2196-99.
(42A) Product identity was readily apparent from the distinct 3-bond phosphorus-hydrogen coupling constant of 8.2 Hz for the two different types of benzylic resonances in the 1H NMR spectra with an integrated ratio of 2:1. 1H NMR (CDCl3/CD3CN (1:1), 300 MHz) ∂7.30 (m, 10H, 2(C6H5)), 6.87 (s, 2H, C6H2), 4.95 (d, J=8.2 Hz, 4H, 2(CH2Ph)), 4.84 (d, J=8.2 Hz, 2H, CH2Ar), 2.13 (s, 6H, 2(CH3).
(43A) For leading references, see: Mccracken; P. G.; Bolton, J. L.; Thatcher, G. R. J. xe2x80x9cCovalent Modification of Proteins and Peptides by the Quinone Methide from 2-tert-Butyl-4,6-dimethylphenol: Selectivity and Reactivity with Respect to Competitive Hydration,xe2x80x9d J. Org. Chem. 1997, 62, 1820-25.
(44A) Acylation of 3,5-dimethyl-4-hydroxybenzaldehyde followed by NaBH4 reduction and phosphorylation of the benzyl alcohol afforded 9 (FIG. 4). (a) The phosphorylation reaction was a modification of: Silverberg, J. J.; Dillon, J. L.; Vemishetti, P. xe2x80x9cA Simple, Rapid and Efficient Protocol for the Selective Phosphorylation of Phenols with Dibenzylphosphite,xe2x80x9d Tetrahedron Eett. 1996, 37, 771-74. (b) A recent paper reports on the hydrolytic stability of benzyltrialkyphosphates and the effects of various substituents on the phenyl ring: Meier, C.; Habel, L. W.; Baizarim, J.; De Clercq, E. xe2x80x9c5xe2x80x2,5xe2x80x2-Di-O-nucleosyl-Oxe2x80x2-benzylphosphotriesters as potential Prodrugs of 3xe2x80x2-Azido-2xe2x80x2,3xe2x80x2-dideoxythymidine-5xe2x80x2-monophosphate,xe2x80x9d Liebigs Ann 1995, 2203-08
(45A) Studies of various electron-donating and electron-withdrawing substituents on the quinone methide ring and at the benzylic methylene have demonstrated their influence on quinone methide formation, reactivity and product stability: (a) Bolton, J. L.; Comeau, E.; Vukomanovic, V. xe2x80x9cThe Influence of 4-Alkyl Substituents on the Formation and Reactivity of 2-Methoxy-Quinone Methides: Evidence That Extended xcfx80-Conjugation Stabilizes the Quinone Methide Formed From Eugenol,xe2x80x9d Chem-Biol. Interactions 1995, 95, 279-90. (b) Thompson, D. C.; Perera, K. xe2x80x9cInhibition of Mitochondrial Respiration by a Para-Quinone Methide,xe2x80x9d Biochem. Biophys. Res. Commun. 1995, 209, 6-11. (c) Lycka, A.; Snobl, D.; Koutek, B.; Pavlickova, L.; Soucek, M. xe2x80x9c13C NMR Study of Substituted Quinone Methides. 2- and 2,6-Substituted Fuchsones,xe2x80x9d CoIl. Czech. Chem. Commun. 1981, 46, 1775-87. (d) Velek, J.; Koutek, B.; Musil, L.; Vasickova, S.; Soucek, M. xe2x80x9cIR Spectra of Some Quinone Methides. A Study of the ortho-Effect,xe2x80x9d Coll. Czech. Chem. Commun. 1981, 46, 873-83.
(46A) (a) Tumbull, K. D. xe2x80x9cpara-Quinone Methides: Chemistry and Exploitation as Intermediates for the Intramolecular Formation of Carbon-Carbon Bonds ald Investigations into the Chemistry and Synthesis of Neolignans Via a Proposed Intermediate in Their Biosynthesis,xe2x80x9d Ph.D. Dissertation, University of California, Riverside, 1991. (b) Angle, S. R.; Turnbull, K. D. xe2x80x9cPara-Quinone Methide Initiated Cyclization Reactions,xe2x80x9d J. Am. Chem. Soc. 1989, 111, 1136.
(47A) A Fries rearrangement on 2,4-dimethylphenol followed by benzylation of the phenol, a Baeyer-Villiger oxidation, and saponification of the intermediate acetate on workup afforded the required mono-protected catechol. A substitution reaction with bromoacetic acid provided the intermediate carboxylic acid which was converted to the desired etser derivatives through carbodiimide activation in the presence of the required alcohol. The dimethylamide derivative was produced directly from addition of bromoacetamide followed by methylation. Hydrogenolysis provided the various derivatives of 11 shown in the table of scheme 3 for oxidation to the desired quinone methides.
(48A) Reliable Biopharmaceuticals, St. Loius, Mo., has generously donated multi-milligram quantities of TpT (with and without protecting groups) and CpA (with and without protecting groups) for the purpose of these studies.
(49A) Wakselman, M. xe2x80x9c1,4- and 1,6-Eliminations from Hydroy- and Amino substituted Benzyl Systems: Chemical and Biochemical Applications,xe2x80x9d Nouv. J. Chim. 1983, 7, 439-47.
(50A) (a) Kanamathareddy, S.; Gutsche, C. D. xe2x80x9cCalixarenes: Selective Functionalization and Bridge Building,xe2x80x9d J. Org. Chem. 1995, 60, 6070-75. (b) Alam, I.; Sharma, S. K.; Gutsche, C. D. xe2x80x9cThe Quinonemethide Route to Mono and Tetrasubstituted Calix[4]arenes,xe2x80x9d J. Org. Chem. 1994, 59, 3716-20. (c) Note that even aniline has been eliminated to produce quinone methides: Angle, S. R.; Yang, W. xe2x80x9cSynthesis and Chemistry of a Quinone Methide Model for Anthracycline Antitumor Antibiotics,xe2x80x9d J. Am. Chem. Soc. 1990, 112, 4524-28.
(51A) Quinone methides have been generated by laser flash photolysis (266 nm) of phenolic benzyl alcohols and the UV of the transient intermediate was presumed to be the quinone methide. These are differentiated from caged quinone methide precursors which can be irradiated at wavelengths outside 350 nm in order to be useful in the presence of biological molecules: Diao, L.; Yang, C.; Wan, P. xe2x80x9cQuinone Methide Intermediates from the Photolysis of Hydroxybenzyl Alcohols in Aqueous Solution,xe2x80x9d J. Am. Chem. Soc. 1995, 117, 5369-70.
(52A) This allows transmission of  greater than 350 nm wavelength which will prevent absorbance of biological aromatics.
(53A) Fleming, S. A.; Jensen, A. W. xe2x80x9cSubstituent Effects on the Photocleavage of Benzyl-Sulfur Bonds. Observation of the xe2x80x98Meta-Effectxe2x80x99xe2x80x9d J. Org. Chem. 1996, 61, 7040-44, and references therein.
(54A) (a) Dimethoxybenzoin: Pirrung, M. C.; Shuey, S. W. xe2x80x9cPhotoremovable Protecting Groups for Phosphorylation of Chiral Alcohols. Asymmetric Synthesis of Phosphotriesters of (xe2x88x92)-3xe2x80x2,5xe2x80x2-Dimethoxybenzoin,xe2x80x9d J. Org. Chem. 1994, 59, 3890-97. (b) Sheehan, J. C.; Wilson, R. M.; Oxford, A. W. xe2x80x9cThe Photolysis of MethoxySubstituted Benzoin Esters. A Photosensitive Protecting Group for Carboxylic Acids,xe2x80x9d J. Am. Chem. Soc. 1971, 93, 7222-28. (c) a-Methyl-4,5-dimethoxy-2-nitrobenzyl: Marriott, G. xe2x80x9cCaged Protein Conjugates and Light-Directed Generation of Protein Activity: Preparation, Photoactivation, and Spectroscopic Characterization of Caged G-Actin Conjugates,xe2x80x9d Biochemistry 1994, 33, 9092-97. (d) a-Carboxy-2-nitrobenzyl: Gee, K. R.; Wieboldt, R.; Hess, G. P. xe2x80x9cSynthesis and Photochemistry of a New Photolabile Derivative of GABA. Neurotransmitter Release and Receptor Inactivation in the Microsecond Time Region,xe2x80x9d J. Am. Chem. Soc. 1994, 116, 8366-67.
(55A) For reviews of photolabile protecting groups, see: (a) Adams, S. R.; Tsien, R. Y. xe2x80x9cControlling Cell Chemistry with Caged Compounds,xe2x80x9d Annu. Rev. Physiol. 1993, 55, 755.84. (b) Gurney, A. M.; Lester, H. A. xe2x80x9cLight Flash Physiology with Synthetic Photosensitive Compounds,xe2x80x9d Physiol. Rev. 1987, 67, 583-617. (c) Pillai, V. N. R. xe2x80x9cPhotoremovable Protecting groups in Organic Synthesis,xe2x80x9d Synthesis 1980, 1-26.
(56A) (a) Derivatives with dimethyl substituents and a benzylic methyl (R, Rxe2x80x2, Rxe2x80x3=Me) were prepared by acylation of 2,6-dimethylphenol followed by a Fries rearrangement to the 4-methylketone derivative followed by protection with o-nitrobenzyl bromide and NaBH4 reduction to the benzyl alcohol derivative. The benzyl alcohol was converted to the acetate, trifluoromethyl acetate, bromide or chloride through literature methods. (b) Derivatives with no substituents (R, Rxe2x80x2, Rxe2x80x3=H) were made by protection of 4-hydroxybenzaldehyde with o-nitrobenzyl bromide and reductive amination with the desired dialkylamine. (c) The methoxy substituted derivative (R, Rxe2x80x3=H, Rxe2x80x2=OMe) were made by protecting vanillin with o-nitrobenzyl bromide, NaBH4 reduction and conversion to the chloride with N-chlorosuccinimide. (d) Dimethyl substituted derivatives with no benzylic alkyl group (R, Rxe2x80x2=Me, Rxe2x80x3=H) were prepared from protection of 3,5-dimethyl-4-hydroxybenzaldehyde with the alcohol of the desired photolabile protecting group in the presence of triphosgene to afford the carbonate protected benzaldehyde. NaBH4 reduction and conversion to the acetate or chloride afforded the desired products.
(57A) Besides the experiment reported (scheme 5), we have run several experiments with guanylated octopamine (structure at right) trying to induce quinone methide formation via dehydration. This has been attempted in refluxing TFA with no sign of any type of reaction. We have also obtained preliminary results on the benzylic chlorination of this compound in DMF with phosphoryl chloride. NMR evidence suggests quantitative conversion to the benzyl chloride with no cyclization of the guanidine. Treatment of this intermediate with NaOH resulted in apparent xcex2-elimination to the styrene, but no sign of cyclization was evident by 1H NMR.
(58A) Athough the possibility for this cyclization is readily apparent, a thorough examination of the natural product, synthetic, and medicinal literature has thus far failed to turn up an example of a cyclization such as could occur in any of our proposed ATAR systems (a 3 to 7-exo-tet or trig depending on which system and whether the quinone methide precursor or the quinone methide is cyclizing (see A, B, or C in section 4.3)). Although there are limited examples of guanidine SN2 or conjugate addition reactions, these require deprotonation under strong basic conditions or electrophilic activation under acidic conditions [For a recent example of an SN2 reaction see: Vaidyanatban, C.; Zalutsky, M. R. xe2x80x9cA New Route to Guanidines rom Bromoalkanes,xe2x80x9d J. Org. Chem. 1997, 62, 4867-69.]. Guanidine condensation reactions are well precedented and a useful means for making heterocyclic structures and in natural product synthesis and generally require acid or base catalysis to drive the reactions [For leading references, see: (a) ref 35. (b )Yamamoto, Y.; Kojima, S. xe2x80x9cSynthesis and Chemistiy of Guanidine Derivatives,xe2x80x9d in The Chemistry of Amidines and Amidates, Patai, S.; Rappoport, Z., Eds.; John Wiley and Sons: N.Y. 1991, 485-526. (c) Berlinck, R. G. S. xe2x80x9cNatural Guanidine Derivatives,xe2x80x9d Nat. Prod. Rep. 1996, 377-409.].
(59A) One example of a natural product which has been isolated which has the potential to undergo a 5-exo-trig cyclization was found. This is martinelline; (Witherup, K. M.; Ransom, R. W.; Graham, A. C.; Bernard, A. M.; Salvatore, M. J.; Lumma, W. C.; Anderson, P. S.; Pitzenberger, S. M.; Varga, S. L. xe2x80x9cMartinelline and Martinellic Acid, Novel G-Protein linked Receptor Antagonist from the Tropical Plant Martinella iquitosensis (Bignoniaceae),xe2x80x9d J. Am. Chem. Soc. 1995, 117, 6682-85.). Athough this was not specifically sought out, the natural product was isolated as the 3xc3x97TFA salt and stable to the extraction, characterization and assaying processes. A small amount of the correponding acid which would result from cleavage of the ester bond was also present; but the authors were uncertain if it was an artifact or natural. It was reported as a natural product.
(60A) This is definitively stated as all the components produced in the reaction mixture have been independently characterized by 1H NMR, and there is no visible sign of any new benzylic resonances, or ethyl resonances which would identify the addition of the ethyl guanidine to the quinone methide.
(61A) It is believed that cyclization will not be a concern in the ATAR as long as the guanidine is protonated. Guanidine deprotection will be the final step in our synthetic protocol and will result in the protonated guanidinium ATAR which will remain under aqueous conditions for all uses.
(62A) Bernatowicz, M. S.; Wu, Y.; Matsueda, G. R. xe2x80x9c1H-Pyrrazole-1-carboxamidine Hydrochloride: An Attractive Reagent for Guanylation of Amines and Its Application to Peptide Synthesis,xe2x80x9d J. Org. Chem. 1992, 57, 2497-502.
(63A) lodination of phthalimide-protected octopamine followed by a Stille reaction with tetramethyltin has been accomplished in approx. 98% yield for the two steps.
(64A) Two approaches are underway: (1) Condensation of nitromethane with benzyl-protected 3,5-dimethyl-4-hydroxy benzaldehyde followed by in situ acylation of the resulting alcohol according to a literature preparation (Wollenberg, R. H.; Miller, S. J. xe2x80x9cNitroalkane Synthesis. A Convenient Method for Aldehyde Reductive Nitromethylation,xe2x80x9d Tetehedron Lett. 1978, 35, 3219-22.). Hydrogenation will debenzylate and reduce the nitro to the amine which will be guanylated using standard procedures and protected as the nitrobenzyl carbonate. (2) Diiodination of octopamine followed by Stile methylation, guanylation, protection of the phenol as the nitrobenzyl carbonate followed by chlorination of the benzylic alcohol. All of these steps have already been accomplished in related systems.
(65A) Yamamoto, Y.; Kojima, S xe2x80x9cSynthesis and Chemistry of Guanidine Derivatives,xe2x80x9d in The Chemistry of Amidines and Amidates, Patai, S.; Rappoport, Z., Eds.; John Wiley and Sons: N.Y. 1991, 485-526.
(66A) Cliffe, I. A. xe2x80x9cFunctions Containing an Iminocarbonyl Group and any Element Other Than a Halogen or a Chalcogen,xe2x80x9d in Comprehensive Organic Functional Group Transformations, Gilchrist, T. L., Ed.; Pergamon: U.K. 1995, 639-75.
(67A) The potential for a phenyl substituent to increase intercalation is realized. As the phenyl group itself is not very polar in nature, this should not be a favored interaction.
(68A) Molecular modeling has not been carried out on these systems due to the highly flexible directionality and tautomerizing nature of hydrogen bonding.
(69A) Although we have been using more traditional brominations, we will try a new procedure which is reported to give 5% or less of the dibromo-product with phenols. Mashraqui, S. H.; Mudaliar, C. D.; Hariharasubrah-manian, H. xe2x80x9c4,4-Dimethyl-3-methylpyrazol-5-one: New Applications for Selective Monobromination of Phenols and Oxidation of Sulfides to Sulfoxides,xe2x80x9d Tetrahedron Lett. 1997, 38, 4865-68.
(70A) Barluenga, J.; Garcia-Martin, M. A.; Gonzalez, J. M.; Clapes, P.; Valencia, G. xe2x80x9clodination of Aromatic Residues in Peptides by Reaction with IPy2BF4,xe2x80x9d J. Chem. Soc. Chem. Commun. 1996, 1505-06.
(71A) Scott, W. J. xe2x80x9cThe Stille Reaction,xe2x80x9d Org. React. 1997 50, 1-652.
(72A) Pappas, J. J.; Keaveney, W. P.; Gancher, E.; Berger, M. xe2x80x9cA New and Convenient Method for Converting Olefins to Aldehydes,xe2x80x9d Tetrahedron Lett 1966, 4273-78.
(73A) Borch, R. F.; Bernstein, M. D.; Durst, H. D. xe2x80x9cThe Cyanohydridoborate Anion as a Selective Reducing Agent,xe2x80x9d J. Am. Chem. Soc. 1971, 93, 2897-904.
(74A) De Maijere, A.; Meyer, F. E. xe2x80x9cFine Feathers Make Fine Birds: The Heck Reaction In Modern Garb,xe2x80x9d Angew. Chem. Int. Ed. EngI. 1994, 33, 2379-411.
(75A) Pasto, D. J.; Taylor, R. T. xe2x80x9cReduction with Diimide,xe2x80x9d Org. React. 1991, 40, 91-155.
(76A) Danishefsky, S. xe2x80x9cSiloxy Dienes in Total. Synthesis,xe2x80x9d Acc. Chem. Res. 1981, 14, 400-406.
(77A) Sibi, M. P.; Stessman, C. C.; Schultz, J. A.; Christensen, J. W.; Lu, J.; Marvin, M. xe2x80x9cA Convenient Synthesis of N-Methoxy-N-methylamides From Carboxylic Acids,xe2x80x9d Synth. Commun. 1995, 25, 1255-64.
(78A) Thompson, C. M.; Green, D. L. C. xe2x80x9cRecent Advances in Dianion Chemistry,xe2x80x9d Tetrahedron, 1991, 47, 4223-85.
(79A) Sibi, M. P. xe2x80x9cApplications of N-Methoxy-N-methylamides in Synthesis,xe2x80x9d Org. Prep. Proced. Int. 1993, 23, 15.
(80A) Danishefsky, S.; Yan, C.-F.; Singh, R. K.; Gammill, R. B.; McCurry, P. M.; Fritsch, N.; Clardy, J. xe2x80x9cDerivatives of 1-Methoxy-3-trimethylsilyloxy-1,3-butadiene for Diels-Alder Reactions,xe2x80x9d J. Am. Chem. Soc. 1979, 101, 7001-008.
(81A) Danishefsky has examined the cycloaddition reactions of sterically bulky dienes similar to 28 with methyl substituents at the 2- and 4-positions.80 Despite the increased sterics, reaction through the s-cis conformation is still a facile process. The same researchers demonstrated the use of dimethyl allene-1,3-dicarboxylate (29) as a dienophile in reactions with Danishefsky""s diene to afford high regioselectivity and yield of a benzene derivative with a substitution pattern similar to our target.80 
(82A) Euranto, E. K. in The Chemistry of Carboxylic Acids and Esters, Patai, S., Ed.; Interscience Publishers: N.Y. 1969, pp. 505-588.
(83A) This will be similar to Overman""s tandem Curtius-carbamylation reaction: Overman, L. E.; Taylor, G. F.; Petty, C. B.; Jessup, P. J. xe2x80x9ctrans-1-N-Acylamino-1,3-dienes: Preparation From Dienoic Acid,xe2x80x9d J. Org. Chem. 1978, 43, 2164-67.
(84A) (a) Sulfonamides are the most stable amine protecting group; however, unlike most sulfonamides, this derivative is readily deprotected with fluoride ion. This will be particularly imperative as it will be the final protecting group removed in the synthesis of the fully functionalized ATAR. Weinreb, S. M.; Demko, D. M.; Lessen, T. A. xe2x80x9cxcex2-trimethylsilylethanesulfonyl Chloride (SES-Cl): A new Reagent for Protection of Amines,xe2x80x9d Tetrahedron Lett 1986, 27, 2099-2102. (b) Alternatively, a more acid labile sulfonamide protecting group (Pmc) will be used: Ramage, R.; Green, J.; Blake, A. J. xe2x80x9cAn Acid Labile Arginine Derivative for Peptide Synthesis: NG-2,2,5,7,8-Pentamethylchroman-6-sulfonyl-L-argirine,xe2x80x9d Tetrahedron 1991,47, 6153-70.
(85A) Hagihara, M.; Schreiber, S. L. xe2x80x9cReassignment of Stereochemistry and Total Synthesis of Thrombin Inhibitor Cyclotheonamide B,xe2x80x9d J. Am. Chem. Soc. 1992, 114, 6570-71.
(86A) Chem. Pharm. Bull. 1984, 33, 1016.
(87A) Sharp, M. J.; Cheng, W.; Snieckus, V. xe2x80x9cSynthetic Connections to the Aromatic Directed Metallation Reaction. Functionalized Aryl Boronic Acids by Ipso Borodesilylation. General Syntheses of Unsymmetrical Biphenyls and m-Terphenyls,xe2x80x9d Tetrahedron Lett. 1987, 28, 5093-96.
(88A) The synthesis would be accomplished as follows is shown in FIG. S9A.
(89A) The guanidine will be deprotected on a derivative having an ester for lactonization.
(90A) The products ofthese reactions will be useful in all future studies for correlation with ATAR alkylated products which can be digested down to the enzymatically protected trialkylphosphate dinucleotide components.
(91A) If necessary, product analysis will take advantage of procedures used for purifying hydrophobic methylphosphonate modified oligonucleotides: Lin, S.-B.; Chang, G.-W.; Teb, G.-W. Lin, K.-I.; Au, L.-C. xe2x80x9cA Simple and Rapid Method for Purification of Oligodeoxyribonucleoside Methylphosphonates,xe2x80x9d Biotechniques 1993, 14, 795-98.
(92A) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning 2nd Ed. (Cold Springs Harbor Laboratory, Cold Springs Harbor, N.Y., 1989).
(93A) The higher the degree of alkylation, the slower the oligo should migrate; or if the polarity is reversed, the faster they will migrate. This will be quantified by densitometry of the autoradiography to allow determination of the degree of alkylation.
(94A) Alternatively, HPLC will also allow resolution and quantification of the relative degree of alkylation.
(95A) i.e., Is there a difference in degree of alkylation at the ends or the middle of the oligonucleotide?
(96A) Diastereomers afford differing 31P resonance signals. An NMR nano-probe would greatly facilitate these analyses, and finding for such an upgrade will be sought. (a) Lxc3x6schner, T.; Engels, J, W. xe2x80x9cDiastereomeric Dinucleoside-methylphosphonates: Determination of Configuration with the 2-Dxcx9cNMR ROESY Technique,xe2x80x9d Nuleic Acids Res. 1990, 18,5083-88. (b) Summers, M. F.; Powell, C.; Egan, W.; Byrd, R. A.; Wilson, W. D.; Zon, G. xe2x80x9cAlkyl Phosphotriester Modified Oligodeoxyribonucleotides. VI. NMR and UV Spectroscopic Studies of Ethyl Phosphotriester (Et) Modified Rp-Rp and Sp-Sp Duplexes, {d[GGAA(Et)TTCC]}2xe2x80x9d Nucleic Acids Res. 1986, 14, 7421-37. (c) Pramanik, P.; Kan, L. xe2x80x9cNMR Study of the Effect of Sugar-phosphate Backbone Ethylation on the Stability and Conformation of DNA Double Helix,xe2x80x9d Biochemistry 1987, 26, 3807-12.
(97A) Oligonucleotides will be synthesized on an automated synthesizer and purified according to standard protocols: (a) Beaucage, S. L.; Caruthers, M. H. Tetrahedron Lett. 1981, 22, 1859. (b) Sinha, N. D.; Biemat, J.; McManus, J.; Koster, H. Nucleic Acids Res. 1984, 12, 4539-57.
(98A) The commercially available 1,12-dodecanediol will be protected as the 4,4xe2x80x2-dimethoxytrityl ether (Khorana, H. G. Pure AppI. Chem. 1968 17, 349.) and converted to the synthesizer-ready phosphoramidite according to standard protocol (Gait, M. J., Ed., Oligonucleotide Synthesis, A Practical Approach, IRL Press: New York; 1990, pp. 41-45.
(99A) Based on modeling estimates, an initial linker will have 16 atoms from the 5xe2x80x2-phosphate oxygen of the oligonucleotide to the benzyl ring of the reagent.
(100A) Oakley, M. G.; Turnbull, K. D.; Dervan, P. R. xe2x80x9cSynthesis ofa Hybrid Protein Containing the Iron-Binding Ligand of Bleomycin and the DNA-Binding Domain of Hin,xe2x80x9d Bioconjugate Chem. 1994, 5, 242-47.
(101A) Bergeron, R. J.; McManis, J. J. J. Org. Chem. 1988, 53, 3108. This can be accomplished without hydrolyzing a methyl ester, so the oligonucleotide attachment will remain unharmed.
(102A) Telser, J.; Cruickshank, K. A.; Morrison, L. E.; Netzel, T. L. xe2x80x9cSynthesis and Characterization of DNA Oligomers and Duplexes Containing Covalently Attached Molecular Labels: Comparison of Biotin, Fluorescein, and Pyrene Labels by Thermodynamic and Optical Spectroscopic Measurements,xe2x80x9d J. Am. Chem. Soc. 1989, 111, 6966-76.
(103A) Han, H.; Dervan, P. R. xe2x80x9cVisualization of RNA Tertiary Structure by RNA-EDTA.Fe(II) Autocleavage: Analysis of tRNAPhe with Uridine-EDTA.Fe(II) at Position 47,xe2x80x9d Proc. NatI. Acad. Sci. USA 1994, 91, 4955-59
(104A) Telser, J.; Cruickshank, K. A.; Schanze, K. S.; Netzel, T. L. xe2x80x9cDNA Oligomers and Duplexes Containing a Covalently Attached Derivative of Tris(2,2xe2x80x2-bipyridine)ruthenium(II): Synthesis and Characterization by Thermodynamic and Optical Spectroscopic Measurements,xe2x80x9d J. Am. Chem. Soc. 1989, 111, 7221-26.
(105A) The standard deprotection methods will be tested in model systems first (ammonium hydroxide), and if necessary, more mild deprotection methods have been developed for solid-support oligonucleotide deprotection and cleavage of alkali-labile oligonucleotides such as with 5% K2CO3 in MeOH.12c A more labile oxallyl-CPG solid-support linkage can be used with this deprotection methodology.12d Alternatively, in conjunction with the oxallyl-CPG, an allyloxy protection scheme can be used throughout the oligonucleotide for mild catalytic palladium deprotection,12b which the carbonate and ester will be stable towards.
(106A) A post-synthetic methodology could also be used to functionalize the deprotected and cleaved, modified oligonucleotide in solution.
(107A) The absorbance ratio at 260 nm (for DNA) versus 350 nm (for the reagent).
(108A) The protocol for triple helix formation and analysis by affinity cleavage is very well developed, see: (a) Greenberg, W. A.; Dervan, P. B. xe2x80x9cEnergetics of Formation of Sixteen Triple Helical Complexes Which Vary at a Single Position Within a Purine Motif,xe2x80x9d J. Am. Chem. Soc. 1995, 117,5016-22.
(109A) Kennard, O.; Hunter, W. N. xe2x80x9cSingle-Crystal X-Ray Diffraction Studies of Oligonucleotides and Oligonucleotide-Drug Complexes,xe2x80x9d Angew. Chem. Int. Ed. Engl. 1991, 30, 1254-77.
(110A) Professor Watkins, Chair of Journalism, has suggested the development of a workshop for professional journalists on xe2x80x9creporting science.xe2x80x9d She described such a workshop as a valuable service to the profession and that the Arkansas Press Association would likely endorse it.
(111A) Nadine Baum Teaching Grant
112A) Course and Curriculum Development
113A) I have received a great deal of help and advice in developing this course from Mrs. Ricki Lewis, a free-lance science journalist who writes for a variety of professional scientific publications. She has expressed interest in being a part of such instruction.
114AA) Although there were only two of these presentations during the course, many written responses at the end of the course demonstrated the appeal of these presentations to the students.
Introduction
The prolific use of reactive quinone methide intermediates in organic and medicinal chemistry1B,2B warrants further optimization of their stability, reactivity, and chemoselectivity for expanding applications. In developing a research program around the application of quinone methides to drug delivery and biomolecular labeling,2B,3B we are studying various ways to control quinone methide formation for its use in biorelevant reactions.4B 
One goal of this research has been to develop a mild method for the latent formation of reactive quinone methide derivatives. Controlled formation of this alkylating intermediate would allow much higher target specificity. The most common method to form para-quinone methides for bioalkylations is through 1,6-elimination of a benzylic leaving group from the corresponding phenol (2 to 3,FIG. S1B).3 For latent formation of quinone methide 3, we considered the use of a protecting group which could be efficiently removed under biorelevant conditions. The reaction sequence would be initiated by removing the protecting group from the quinone methide precursor 1 to form the phenol 2 (FIG. S1B). Subsequent elimination of the leaving group produces quinone methide 3. The ability to initiate the latent formation of the quinone methide will improve selectivity in alkylation of the target bionucleophile to afford alkylated product 4.
The competitive pathway available to quinone methide precursor 1 in aqueous systems is hydrolysis to form benzyl alcohol 5. This has resulted in serious limitations to the use of quinone methides in biomolecular alkylation applications.3 The rate of this competing hydrolysis pathway can be somewhat moderated by appropriate modification of the quinone methide precursor.
Meier and co-workers have conducted hydrolytic stability studies on the effect of ring substitution in benzylic trialkylphosphates and concluded that substitution by electron donating groups at the 4 position increases the potential for hydrolysis of a benzylic trialkylphosphate.5 Most notable, in relation to our work, is that they were unable to isolate a benzylic trialkylphosphate having a 4-methoxy substituent. Widlanski and coworkers showed that the addition of a 2-nitro group to the ring (R=NO2,FIG. S1B) improved the hydrolytic stability of a benzylic compound capable of forming a quinone methide through 1,6-elimination.6 The stabilizing effect of a 2-nitro group allowed access to the quinone methide precursor having a bromide or chloride leaving group at the benzylic position (LG=Br or Cl,FIG. S1B).
Desiring a general, mild method for quinone methide formation under aqueous conditions, we investigated photolytic initiated 1,6-elimination reactions. Photolytic initiation at xe2x89xa7350 nm wavelengths was considered ideal for a mild generation of a latent quinone methide for biomolecular labeling purposes. Photolabeling techniques have proven widely applicable in biochemistry.7B Our interest was in the use of 2-nitrobenzyl and xcex1-methylnitroveratryl as photolabile protecting groups to be used for latent formation of quinone methides. The 2-nitrobenzyl protecting group has found widespread use in bioorganic and synthetic applications.5B,8B,9B The xcex1-methylnitroveratryl protecting group is based on the 2-nitrobenzyl group but has more stable photolysis byproducts.10A 
We wish to report the results of work in our laboratory on the hydrolytic stabilization of quinone methide precursors having benzylic leaving groups, and the latent generation of quinone methides via photolytic initiation. Based on previous investigations,5A an ether-linked protecting group on the quinone methide precursor (PG,FIG. S1B) was expected to be unstable towards hydrolysis. The use of an electron withdrawing group as a linker to the protecting group was expected to afford stability. Therefore, we desired to investigate the stabilizing effect of a carbonate linked photolabile protecting group versus an ether linkage in the quinone methide precursor. Our investigations required the synthesis of a series of quinone methide precursors with ether- and carbonate-linked photolabile protecting groups and bromide, chloride or fluoride leaving groups.
Results and Discussion
The synthesis of a series of related quinone methide precursors for our investigations was accomplished as outlined in FIG. T1B. Commercially available 4-hydroxy-3,5-dimethylbenzaldehyde (6) was protected as either the ether or the carbonate (7) by using the corresponding benzylbromide or chioroformate, respectively (FIG. T1B).xe2x80x9d The bromide of the 2-nitrobenzyl group is commercially available while that of the xcex1-methylnitroveratryl is available in one step from the alcohol.12B The necessary xcex1-methylnitroveratryl alcohol was readily available upon nitration of the corresponding veratryl aldehyde10B followed by methyl acyl addition with trimethylaluminum.13B The cliloroformates required for carbonate formation are derived from the corresponding alcohols.14B After appropriate protection of 6, aldehyde 7 was then reduced to alcohol 8 with sodium borohydride.15B The resulting alcohol was transformed into the desired halide (9). The chloride is readily prepared using triphosgene.16B 
ai) 6, 2-nitrobenzylbromide, K2CO3, DMF, r.t., 2 h. ii) 6, a-methylnitroveratrylbromide, K2CO3, DMF, r.t., 44 h. iii) 6, 2nitrobenzylchloroformate, K2CO3, DMF, r.t., 1.5 h. iv) 6, a-methylnitroveratrylalcohol, triphosgene, py., CH2Cl2, xe2x88x9242 C-r.t., 16h.b NBE=2-Nitrobenzylether; MNVE=cc-Methylnitroveratrylether; NBC=2-Nitrobenzylcarbonate; MNVC=xcex1-Methylnitroveratrylcarbonate.c NaBH4, EtOH.d i) 8, triphosgene, py., CH2Cl2, r.t.
After forming each of the desired quinone methide precursors (9), they are being monitored for hydrolytic stability in various concentrations of water/acetonitrile at various temperatures (table 2). Solutions of the substrates (5 mM) in 5%, 10%, 33% and 50% water/acetonitrile at 25xc2x0 C. are being monitored using capillary GC and 1H NMR analysis. Solutions of the substrates which are appropriately stable in 50% water/acetonitrile are then being monitored at temperatures of 25xc2x0 C., 37xc2x0 C. and 50xc2x0 C. by capillary GC analysis. FIG. T2B shows the results of the hydrolysis half-lives for the various quinone methide precursors.
Preliminary results indicate that the ether-protected quinones methide precursors having a chloride leaving group at 22xc2x0 C. in 33% D2O/CD3CN have a significantly shorter hydrolysis half-life compared to their carbonate-protected analogs. Additionally, a change in the protecting group from the 2-nitrobenzyl to the xcex1-methylnitroveratryl resulted in an increase in the hydrolysis half-life of the quinone methide precursors having a chloride leaving group. The xcex1-methylnitroveratryl carbonate protected substrate with a fluoride leaving group is expected to be the most stable to hydrolysis. The leaving groups within each class of protecting group (ether or carbonate) are anticipated to have hydrolysis half-lives in the order fluorine greater than chlorine greater than bromine. A change in the linking group from ether to carbonate is expected to result in a marked increase in the hydrolysis half-life regardless of the protecting group or leaving group used. This is believed to be the result of the electron withdrawing capacity of the carbonate group compared to the ether group.